Reserarchers found compound for the treatment of melioidosis

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Melioidosis is a tropical disease that claims an estimated 90,000 lives worldwide each year.

There is no vaccine, and current treatments are hampered by the ability of the bacterium that causes the disease to resist even the strongest antibiotics.

Hardy and lethal, that bacterium, Burkholderia pseudomallei, is classified by the Centers for Disease Control and Prevention as a potential bioweapon.

UCLA-led research has identified two compounds that, based on tests on human cells and on mice, show potential for treating melioidosis.

One is a widely used drug already approved by the U.S. Food and Drug Administration as an antifungal treatment; the other is a new synthetic antibiotic. The findings, published in the Proceedings of the National Academy of Sciences, represent progress against a disease about which little is known.

“Even among neglected tropical diseases, melioidosis is one of the most neglected, especially when you consider its global burden and lethality,” said senior author Jeff F. Miller, UCLA’s Fred Kavli Professor of NanoSystems Sciences, a professor of microbiology, immunology and molecular genetics, and director of the California NanoSystems Institute at UCLA. “It deserves more attention because of its fascinating biology as well.”

Seen most often in southeast Asia and northern Australia, melioidosis can take many forms, ranging from a lethal bloodstream infection to a chronic infection that mimics other diseases like tuberculosis.

B. pseudomallei can be contracted when contaminated soil or water is inhaled, swallowed or taken in through a wound or cut.

When it enters mammalian cells, the bacterium multiplies and can spread by causing infected cells to fuse with their neighbors.

That capacity to fuse cells together is critical to the bacterium’s lifecycle and its ability to cause disease.

Eventually, complexes of fused cells burst, which can destroy the tissue in which the cells are located.

Working at the California NanoSystems Institute’s robotic screening facility, and using a screening technique they developed, Miller and his colleagues tested a library of more than 220,000 compounds – thousands at a time – for their ability to interrupt the bacterial life cycle.

“The screen we developed would be easy to adapt to other intracellular pathogens, such as Mycobacterium, which causes tuberculosis,” said Christopher Todd French, the paper’s co-corresponding author and a UCLA assistant research professor.

“It captures any compound that inhibits a step along the way from the bacterium getting into the cell, to it growing in the cell, to it spreading to nearby cells.”

Because of the extensive safety precautions required to study B. pseudomallei, the researchers first screened the compounds on a less-dangerous relative, B. thailandensis, to narrow down the list of drugs that warranted further testing.

For each screening, human cells were robotically placed on lab plates, treated with the compounds to be tested, and then infected with the bacterium.

After 20 hours, the scientists used an imaging method called laser scanning cytometry to take high-resolution images of the plates to identify the effects of each compound.

Compounds that disrupted B. thailandensis were then tested against B. pseudomallei and B. mallei, another closely related bacterium that causes glanders, a disease that’s most commonly seen in horses and other animals but can also affect humans. Ultimately, the researchers identified 32 compounds that worked against B. pseudomalleiB. mallei or both.

The team focused on two compounds that were particularly effective against B. pseudomallei and showed minimal toxicity toward host cells.

Both were able to enter the host cell and the bacterium – a significant development because getting compounds to cross both barriers is one of the key challenges in developing new antibiotics.

One of the two was flucytosine, or 5-FC, the antifungal medication already approved by the FDA. Researchers found that, rather than stopping the growth of B. pseudomallei, 5-FC worked by stopping the bacterium from fusing cells together and spreading.

That characteristic places 5-FC among an emerging class of antibiotics called antivirulence drugs, which could potentially be paired with traditional antibiotics – drugs that work by stopping bacteria from growing in the first place.

That one-two punch could attack harmful bacteria more effectively while making antibiotic resistance less likely. Additionally, 5-FC is on the World Health Organization’s Model List of Essential Medicines, meaning that it is available in resource-poor settings where melioidosis is endemic.

“A key priority will be to translate these laboratory findings into clinical impact,” said Dr. Philip Bulterys, the study’s first author and a recent graduate of the UCLA–Caltech Medical Scientist Training Program.

“5-FC already has a well-established safety profile, which should make for a more rapid route to evaluating it as a new potential treatment for melioidosis.”

The other potential new therapy, which the researchers dubbed burkfloxacin, or BFX, is a synthetic molecule related to antibiotics in the fluoroquinolone class, which includes the well-known drug ciprofloxacin.

In the study, it was extremely effective at blocking B. pseudomallei‘s growth.

Studying mice infected with a lethal dose of B. pseudomallei, the scientists found that both 5-FC and BFX performed better than the current standard of care for melioidosis treatment, ceftazidime.


Melioidosis results from infection with the Gram-negative bacterium Burkholderia pseudomallei and has diverse clinical presentations including pneumonia, localised cutaneous lesion, bacteremia without evident focus, osteomyelitis, septic arthritis and severe sepsis with multiple organ abscesses (Wiersinga et al., 2012Wiersinga et al., 2018).

It is endemic in Southeast Asia and northern Australia, with increasing recognition in many other tropical and sub-tropical locations (Limmathurotsakul et al., 2016).

Therapy is divided into an intravenous intensive phase and an oral eradication phase (Dance, 2014Lipsitz et al., 2012).

International guidelines recommend at least 10–14 days intravenous therapy with either ceftazidime or a carbapenem, followed by a minimum of 12 weeks of oral trimethoprim-sulfamethoxazole (Lipsitz et al., 2012).

Amoxicillin-clavulanic acid and doxycycline are considered second line agents (Lipsitz et al., 2012).

There are low rates of adherence to oral eradication therapy in our region, which has led to the progressive lengthening of the duration of intravenous intensive phase therapy (Pitman et al., 2015).

Duration of intravenous therapy recommended in northern Australia is determined by the site and severity of melioidosis, based on the antibiotic duration determining focus as previously defined (Pitman et al., 2015).

For instance, intravenous therapy is recommended for four weeks for complicated pneumonia and deep-seated abscess, at least six weeks for osteomyelitis and eight weeks for neurological melioidosis and arterial infection (Pitman et al., 2015).

This therapy is often given via a Peripheral Inserted Central Catheter (PICC) with an elastomeric infusor device, which has been shown to be safe and effective in an out of hospital program (Huffam et al., 2004).

The duration of oral eradication therapy is typically three months, but this is extended to six months in neurological, arterial and bone melioidosis (Pitman et al., 2015).

The rationale for oral eradication therapy is to prevent relapse of melioidosis (Currie, 2015Dance, 2014).

The northern Australian guideline has been shown to be associated with low rates of relapse, despite frequent poor adherence to oral eradication therapy, and this low relapse rate has been attributed to the longer duration of intravenous therapy (Pitman et al., 2015Sarovich et al., 2014).

It is therefore possible that some patients in our region could have their oral eradication therapy shortened or even avoided (Pitman et al., 2015). However, the risks of oral eradication therapy are not known so the potential benefits of such reduction are not quantified.

The purpose of this study was to determine the incidence of side effects from oral eradication therapy in our patient cohort.

Discussion

Patients in northern Australia who are treated for melioidosis experience high rates of side effects to oral trimethoprim-sulfamethoxazole, which often results in a change of therapy, a reduction in dose, or cessation of therapy. T

rimethoprim-sulfamethoxazole monotherapy has been internationally considered the first line agent for oral eradication therapy since the randomised controlled trial in Thailand found recurrence rate with trimethoprim-sulfamethoxazole alone was non-inferior to trimethoprim-sulfamethoxazole in combination with doxycycline and was associated with fewer side effects (Chetchotisakd et al., 2014).

The discontinuation rate due to an adverse event from the planned 20 weeks of trimethoprim-sulfamethoxazole was 12% in the Thai study (Chetchotisakd et al., 2014). In our study, 3/203 (1.5%) patients had trimethoprim – sulfamethoxazole ceased completely and 47/203 (23.2%) had their antibiotic changed due to an adverse event.

The higher rates seen in our population group may in part reflect more frequent monitoring of complete blood count, renal and liver function, which typically occurs at least monthly in our setting rather than the 4, 12, and 20 week follow up in Thailand (Chetchotisakd et al., 2014).

In addition there has historically been a tendency for higher doses of trimethoprim – sulfamethoxazole in Australia, although more recently the dosing regimen has been aligned globally (Lipsitz et al., 2012).

Nevertheless, from our clinical experience, most adults with melioidosis in our region are commenced on the maximum dose of 320 + 1600 mg and the substantial rates of side effects to oral trimethoprim-sulfamethoxazole seen in this study most likely reflect this high dosing used for melioidosis.

It is interesting to note that the majority of our patients who experienced a side effect were changed to an alternative agent, most commonly doxycycline and in a few incidences, amoxicillin-clavulanic acid. A study from Thailand showed that doxycycline alone was inferior to conventional treatment with chloramphenicol, trimethoprim-sulfamethoxazole, and doxycycline (Chaowagul et al., 1999).

Amoxicillin – clavulanic acid has been similarly shown to be inferior (Rajchanuvong et al., 1995). Despite an earlier study from Darwin also showing higher relapse with doxycycline (Jenney et al., 2001), there was a low rate of recrudescence and relapse rate in the current study despite 53/212 (25.0%) of patients taking doxycycline at some stage during the oral eradication phase. We examined each case of relapse and recrudescence and found recrudescence was associated with osteomyelitis, and relapse was associated with the presence of unrecognized foci or incomplete eradication therapy when there were extensive internal collections.

This likely reflects an inadequacy of source control rather than a failure of the oral antibiotic itself. We postulate that in recent years we may not be seeing the clinical failure with doxycycline and the concomitant acquired doxycycline resistance that occurred in a past era (Jenney et al., 2001) because of the reduction in bacterial burden from the prolonged intravenous therapy now used and the first line preference for trimethoprim-sulfamethoxazole, even if it subsequently requires a change to doxycycline. Unlike in prior times, with our current regimen few patients are exposed to doxycycline when their bacterial burden is high.

Our guideline recommends doxycycline as the preferred second line agent as in our clinical experience patients have often been unable to tolerate the high doses of amoxicillin-clavulanic acid recommended for the treatment of melioidosis (Cheng et al., 2008).

A limitation of this study is its retrospective nature and the definitions used for adverse effects. An adverse effect was defined as a side effect documented in the medical record or outpatient letter, which was attributed to the oral therapy and necessitated either a change or cessation of therapy, or a reduction in dose.

There is a possibility that some side effects may have been omitted in the medical documentation, while attribution of side effects to trimethoprim-sulfamethoxazole may have been incorrect in some cases. Additionally, we did not seek actual completion rate of oral eradication therapy in this study and we were unable to quantify the adherence of individual patients. We also did not audit the dosing prescribed by clinicians in our population.

In conclusion, despite often poor adherence to oral eradication therapy, there was a high rate of adverse effects from trimethoprim-sulfamethoxazole in patients treated for melioidosis in northern Australia.

Dose reduction or a change to doxycycline therapy was common, but in our setting where prolonged intravenous therapy is possible, treatment was still successful in the vast majority of patients.

Given the side effects and low rates of oral therapy completion in our region we emphasise the importance of prolonged intensive phase intravenous therapy and a trimethoprim-sulfamethoxazole dose based on weight.

Further studies are required to identify if there are subsets of patients who may benefit from a reduction in oral therapy duration or even omission of the eradication therapy phase. Ideally randomised comparative studies with analysis of costs and outcomes can help optimise therapy regimens for melioidosis of differing severity and in different geographical circumstances.


More information: Philip L. Bulterys et al. An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1906388116

Journal information: Proceedings of the National Academy of Sciences
Provided by University of California, Los Angeles


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