• View in gallery

    Schematic representation of near-neighbor Burkholderia species and their relatedness to Burkholderia pseudomallei and other major disease-causing species. Relationships are derived from previous phylogenetic trees.2426,43 Lines represent relationships between the species but not genetic distance. Closed circles indicate Burkholderia species included in this study. The species highlighted in bold are implicated in human disease.

  • View in gallery

    Principal component analysis38 of minimal inhibitory concentrations of antibiotics against Burkholderia strains: Burkholderia pseudomallei (Bp), Burkholderia humptydooensis (Bhu), Burkholderia thailandensis (Bth), Burkholderia territorii (Bte), Burkholderia oklahomensis (Bok), and Burkholderia stagnalis (Bst). (A) Analysis includes ceftazidime, co-trimoxazole, meropenem, trimethoprim, rifampicin, doxycycline, tetracycline, and chloramphenicol. (B) Exclusion of chloramphenicol from the analysis. The percentages of the total variance along the principal component 1 (PC1) and PC2 axes are shown in brackets.

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Safer In Vitro Drug Screening Models for Melioidosis Therapy Development

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  • 1 Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia;
  • | 2 Global and Tropical Health Division, Menzies School of Health Research, Darwin, Australia;
  • | 3 Northern Territory Medical Program, Department of Infectious Diseases, Royal Darwin Hospital, Darwin, Australia;
  • | 4 Queensland University of Technology, School of Biomedical Sciences, Institute of Healthy and Biomedical Innovation, and Translational Research Institute, Brisbane, Queensland, Australia

ABSTRACT

Melioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current antibiotic regimens used to treat melioidosis are prolonged and expensive, and often ineffective because of intrinsic and acquired antimicrobial resistance. Efforts to develop new treatments for melioidosis are limited by the risks associated with handling pathogenic B. pseudomallei, which restricts research to facilities with biosafety level three containment. Closely related nonpathogenic Burkholderia can be investigated under less stringent biosafety level two containment, and we hypothesized that they could be used as model organisms for developing therapies that would also be effective against B. pseudomallei. We used microbroth dilution assays to compare drug susceptibility profiles of three B. pseudomallei strains and five nonpathogenic Burkholderia strains. Burkholderia humptydooensis, Burkholderia thailandensis, and Burkholderia territorii had similar susceptibility profiles to pathogenic B. pseudomallei that support their potential as safer in vitro models for developing new melioidosis therapies.

INTRODUCTION

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis,1,2 a neglected tropical disease, with one model estimating up to 165,000 cases and 89,000 deaths per year.1 Mortality rates for infected individuals vary from under 10% in Darwin (Northern Territory, Australia),3 where state-of-the-art intensive care facilities are available, to more than 40% in endemic regions in Southeast Asia, where health resources are more limited.4

Conventional large-spectrum antibiotic classes, such as aminoglycosides (e.g., streptomycin, gentamicin, and kanamycin), early generation β-lactam antibiotics (e.g., penicillin), fluoroquinolones (e.g., ciprofloxacin), and macrolides (e.g., erythromycin), have little effect on B. pseudomallei, which limits treatment options.58 Importantly, environmental isolates and primary B. pseudomallei isolates are almost universally susceptible to the first-line drugs used for melioidosis therapy, ceftazidime, meropenem, and co-trimoxazole.5,9,10 However, the prolonged nature of melioidosis treatment (4–6 months) can lead to acquired antibiotic resistance, which is linked to treatment failure and mortality.9 To overcome both intrinsic and acquired antibiotic resistance, more efficacious therapies for melioidosis are required.1,11

Efforts to develop new treatments for melioidosis are limited by the classification of B. pseudomallei as a risk group three microorganism (i.e., the potential to cause serious human disease) in most countries.1215 This classification restricts its research to laboratories classified as biosafety level three in the United States,16 or the equivalent physical containment three in Australia and New Zealand.17 Burkholderia pseudomallei is also recognized as a tier one biothreat agent on the Center for Disease Control and Prevention Bioterrorism Agent list,15 a classification that further restricts research efforts.18,19

The use of nonpathogenic Burkholderia species as models for B. pseudomallei is a desirable approach for overcoming these limitations. Burkholderia thailandensis2022 and mutant B. pseudomallei strains Bp82 and Bp190 that are avirulent to mice and hamsters23 have been used as model organisms because of their close genetic relationship to B. pseudomallei, but extensive comparison of their susceptibility to antibiotics or other therapeutic compounds has not yet been described. In addition to these established models, we propose that other closely related Burkholderia species that are not implicated in human disease2426 will also be useful model organisms for melioidosis research.

To determine their suitability as model organisms, we characterized the antibiotic susceptibility of five nonpathogenic Burkholderia strains and three B. pseudomallei strains to a panel of antibiotics and drug-like compounds. Here, we demonstrate that B. thailandensis and two newly characterized strains—Burkholderia humptydooensis and Burkholderia territorii—have susceptibility profiles that recapitulate B. pseudomallei susceptibility. The similarity of these profiles supports the utility of nonpathogenic Burkholderia as models for initial melioidosis therapy development.

MATERIALS AND METHODS

Burkholderia strains.

Burkholderia strains used in this study were B. pseudomallei (MSHR10517, MSHR2154, and MSHR1364), B. humptydooensis (MSMB043), Burkholderia oklahomensis (MSMB0175), Burkholderia stagnalis (MSMB049), B. thailandensis (MSMB0608), and B. territorii (MSMB0110). These strains were collected and cultivated from soil or water samples as previously described (Menzies School of Health Research).2729

Antibiotic panel.

Antibiotics that have been included in this study represent the current standard therapeutics for treating melioidosis, ceftazidime, co-trimoxazole, and meropenem5; and other antibiotics used to treat bacterial infections. To produce a wider susceptibility profile, antibiotics with varying levels of efficacy against B. pseudomallei7,3036 were also included (see Table 1).

Table 1

Minimum inhibitory concentrations of antibiotics against Burkholderia strains*

AntibioticMIC (mg/L)MIC (mg/L)§MIC90 (mg/L)
B. humptydooensisB. oklahomensisB. thailandensisB. stagnalisB. territoriiB. pseudomalleiB. pseudomallei
MSMB0430175MSMB0608MSMB049MSMB0110MSHR1364MSHR2154MSHR10517Various isolates from published reports
MelioidosisCeftazidime1–20.31–24221–221–431,322–47,30
Co-trimoxazole2–40.3–212–412220.125–432
Meropenem10.4–2212–2520.40.410.5–431,32
Other antibioticsCefsulodin> 35> 35> 35> 35> 35> 1287
Amoxicillin> 23> 23> 23> 23> 23> 23> 23> 23> 1287,34
Ampicillin> 24> 24> 24> 24> 24> 24> 24> 2412.5–2533
Sulfamethoxazole≥ 168 to > 16> 16> 16> 1632034
Trimethoprim5–91 to > 1912–5195–996434
Rifampicin1371326–53131313138–16358–327,34
Nalidixic acid7–151515> 157> 16> 16> 16> 5033327
Doxycycline0.4–20.3–10.47–140.3–310.4–110.25–332,351–47,34
Tetracycline2–71–282–142 to > 280.4–7222–41.6–3.1330.5–830,34
Chloramphenicol3–105–105–1010–21–52110–2121∼6–203316–327,34
Kanamycin19–37> 37> 37> 37≥ 37> 37> 37> 376434
Gentamicin> 37> 37> 37> 37> 37> 37> 37> 37> 503364–1287,34
Puromycin> 35> 35> 35> 35> 35> 35> 35> 35
Spectinomycin> 32> 32> 32> 32> 32> 32> 32> 32> 6436
Clarithromycin> 48> 48> 48> 48> 4832–6435
Paromomycin> 40> 40> 40> 40> 40> 5033
Streptomycin> 47> 47> 47> 47> 47> 5033

B. humptydooensis = Burkholderia humptydooensis; B. oklahomensis = Burkholderia oklahomensis; B. pseudomallei = Burkholderia pseudomallei; B. stagnalis = Burkholderia stagnalis; B. territorii = Burkholderia territorii; B. thailandensis = Burkholderia thailandensis; MIC = minimal inhibitory concentration.

Antibiotics: Melioidosis, antibiotics currently used for treating melioidosis; Other antibiotics, used for treating bacterial infections.

Minimal inhibitory concentrations were determined from 100% growth inhibition, using broth microdilution of bacteria in growth phase. Concentrations in μM were converted to mg/L. Data represent the MIC (or range) determined from at least three independent experiments (individual MIC data are shown in Supplemental Table S1).

Minimal inhibitory concentration values indicate the minimum concentration required to inhibit 100% of growth.

Minimal inhibitory concentration 90 values indicate the concentration required to inhibit the growth of 90% of the tested population.

Antibiotics with previously reported high MICs for B. pseudomallei and high tested MICs for the near-neighbor strains were not tested against B. pseudomallei strains in this study.

Antibiotic susceptibility profiles.

Antimicrobial susceptibility was tested using a plate-based broth microdilution method as before,37 except that bacteria were incubated at 30°C because they showed better growth at 30°C than at 25°C and 37°C. In brief, assays were conducted in Mueller Hinton broth with bacteria in mid-log-phase growth that were diluted to ∼106 colony-forming unit (CFU)/mL (optical density at 600 nm = 0.001). Serial dilutions of the compounds were then added to the bacteria (final density ∼5 × 105 CFU/mL). The minimal inhibitory concentration (MIC) was determined to be the lowest concentration of compound that inhibited visible bacterial growth 24 hours after treatment. Data represent the MIC determined from a minimum of three independent experiments for each strain (see Supplemental Table S1 for individual MIC data for all replicates). Principal component analyses were performed using the “prcomp” function, and plots were generated using the “ggplot” function of R version 4.0.0.38

Medicines for Malaria Venture (MMV) Pathogen Box compound susceptibility profiles for B. humptydooensis and B. territorii.

We tested the antimicrobial activity of the MMV Pathogen Box containing a library of 400 drug-like compounds.39 Initial antimicrobial susceptibility testing was performed at 20 μM against B. humptydooensis and B. territorii, using the methods described earlier. Ceftazidime (20 μM) was added to each plate as a positive control for 100% growth inhibition.5 We identified a subset of compounds with promising activity (compound ID, molecular weight, molecular formula, and structure of compounds from this subset are provided in Supplemental Table S2). These compounds were serially diluted to determine the MIC toward B. humptydooensis and B. territorii. Data represent the MIC determined from three independent experiments for each strain.

RESULTS AND DISCUSSION

Burkholderia near-neighbor strains.

The Burkholderia genus comprises more than 70 species with varying virulence and pathogenicity,4042 and these species can be divided according to their relationship to either B. pseudomallei (the B. pseudomallei complex) or Burkholderia cepacia (the B. cepacia complex). In the current study, we included three strains of B. pseudomallei and five representative near-neighbor Burkholderia strains of B. thailandensis, B. humptydooensis, B. oklahomensis, B. stagnalis, and B. territorii. The relationship between the nonpathogenic near neighbors and pathogenic Burkholderia species is represented in Figure 1. Genetic distance from B. pseudomallei is shown in Supplemental Table S3 for corresponding near-neighbor isolates from a previous study.43

Figure 1.
Figure 1.

Schematic representation of near-neighbor Burkholderia species and their relatedness to Burkholderia pseudomallei and other major disease-causing species. Relationships are derived from previous phylogenetic trees.2426,43 Lines represent relationships between the species but not genetic distance. Closed circles indicate Burkholderia species included in this study. The species highlighted in bold are implicated in human disease.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 5; 10.4269/ajtmh.20-0248

Antibiotic susceptibility profiles for B. pseudomallei and near neighbors.

The aim of this investigation was to determine whether the antibiotic susceptibility of B. pseudomallei is recapitulated by near-neighbor species. We hypothesize that less pathogenic species with similar antibiotic susceptibility profiles to B. pseudomallei are useful as in vitro models to facilitate initial screening of new therapeutic molecules without the restrictive physical containment requirements required when working with B. pseudomallei.

We determined the susceptibility of the B. pseudomallei and near-neighbor strains to a panel of 20 antibiotics with previously reported MIC90 or MIC values against B. pseudomallei. The MICs (or MIC range) are compared in Table 1 and show a similar overall susceptibility profile for all of the tested strains. Principal component analysis (PCA) of MICs from the eight most potent antibiotics—ceftazidime, co-trimoxazole, meropenem, trimethoprim, rifampicin, doxycycline, tetracycline, and chloramphenicol—revealed that B. humptydooensis, B. thailandensis, and B. territorii display a similar susceptibility to these antibiotics as B. pseudomallei strains MSHR10517, MSHR2154, and MSHR1364 (Figure 2A). The MICs of chloramphenicol for these B. pseudomallei strains may be considered as outliers, as they are at the higher end of the reported MIC range for B. pseudomallei (∼6–20 mg/L). Indeed, removal of chloramphenicol MICs from the PCA analysis resulted in tighter clustering of B. humptydooensis, B. thailandensis, and B. territorii with B. pseudomallei (Figure 2B).

Figure 2.
Figure 2.

Principal component analysis38 of minimal inhibitory concentrations of antibiotics against Burkholderia strains: Burkholderia pseudomallei (Bp), Burkholderia humptydooensis (Bhu), Burkholderia thailandensis (Bth), Burkholderia territorii (Bte), Burkholderia oklahomensis (Bok), and Burkholderia stagnalis (Bst). (A) Analysis includes ceftazidime, co-trimoxazole, meropenem, trimethoprim, rifampicin, doxycycline, tetracycline, and chloramphenicol. (B) Exclusion of chloramphenicol from the analysis. The percentages of the total variance along the principal component 1 (PC1) and PC2 axes are shown in brackets.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 5; 10.4269/ajtmh.20-0248

Minimal inhibitory concentrations for the antibiotics used to treat melioidosis were similar between near-neighbor B. humptydooensis, B. thailandensis, and B. territorii strains; B. pseudomallei strains from this study; and previously reported MICs for ceftazidime (1–2 mg/L), co-trimoxazole (2 mg/L), and meropenem (0.4–1 mg/L) against various B. pseudomallei strains (see Table 1). Minimal inhibitory concentrations for these antibiotics toward B. oklahomensis were slightly less predictive of B. pseudomallei susceptibility, and B. stagnalis MICs were even less predictive, especially for meropenem (MIC 12–25 mg/L).

The activity of other antibiotics used to treat bacterial infection further illustrated the utility of B. humptydooensis, B. thailandensis, and B. territorii as models for B. pseudomallei. Rifampicin had near-identical activity toward these near-neighbor strains and B. pseudomallei (MIC ∼13 mg, see Table 1). Minimal inhibitory concentration ranges for doxycycline were similar between the near neighbors and B. pseudomallei (0.3–3 versus 0.4–1 mg/L), and the near neighbors were equally, or more, susceptible to trimethoprim (MIC 1–9 mg/L) and chloramphenicol (MIC 3–10 mg/L) than the B. pseudomallei strains (MIC 5–9 mg/L and 10–21 mg/L, respectively). Tetracycline susceptibility of the B. pseudomallei strains (MIC 2–4 mg/L) was within the range of MICs observed for B. humptydooensis, B. thailandensis, and B. territorii. The wider range of MICs observed for tetracycline (see Table 1, Supplemental Table S1) may reflect the bacteriostatic mechanism of action; however, this was not observed for other bacteriostatic antibiotics, including doxycycline and trimethoprim.

Kanamycin and nalidixic acid did not inhibit B. pseudomallei at the highest concentration tested (37 mg/L and 16 mg/L, respectively) but exhibited activity at these concentrations against B. humptydooensis, B. oklahomensis, B. thailandensis, and B. territorii. Finally, nine of the antibiotics—amoxicillin, ampicillin, cefsulodin, clarithromycin, gentamicin, spectinomycin, streptomycin, puromycin, and paromomycin—did not have activity against the tested B. pseudomallei and/or near-neighbor strains, consistent with previous reports of inactivity against various B. pseudomallei isolates7,3336 (see Table 1).

These comparative antibiotic susceptibility screens showed that B. pseudomallei near neighbors B. humptydooensis, B. thailandensis, and B. territorii have similar antibiotic susceptibility profiles to those of B. pseudomallei against key melioidosis therapeutics, as well as other antibiotics (see Table 1, Figure 2).

Susceptibility of B. humptydooensis and B. territorii to MMV compounds.

This study is the first report of antibiotic susceptibilities for B. humptydooensis and B. territorii. To further evaluate their suitability as models for predicting B. pseudomallei drug susceptibility, we also tested their susceptibility to 400 drug-like molecules from the MMV Pathogen Box.39 These data allow independent comparison of MMV compound susceptibility of B. humptydooensis and B. territorii to five previously reported B. pseudomallei strains.44

We initially tested MMV compounds at 20 μM (and identified five compounds with activity against B. humptydooensis and B. territorii). Four of these compounds—doxycycline, levofloxacin, rifampicin, and MMV688271—agreed with reported activity toward B. pseudomallei,44 whereas MMV675968 is a newly identified active compound (see Supplemental Table S2 for compound characteristics). Next, we determined the MICs for these compounds against B. humptydooensis and B. territorii (see Table 2). The activities of doxycycline (MIC 0.5–1 mg/L), levofloxacin (MIC 1–6 mg/L), MMV688271 (MIC 4–8 mg/L), and ceftazidime (MIC 2–4 mg/L) against the near-neighbor strains were within 2-fold of their reported MICs against B. pseudomallei (1–3 mg/L, 4–10 mg/L, 6–12 mg/L, and 3–4 mg/L, respectively).44 Minimal inhibitory concentrations for ceftazidime, doxycycline, and rifampicin determined from the MMV compound screen (Table 2) are in close agreement with MICs from the antibiotic susceptibility screen (Table 1). Principal component analysis of the MICs of these three antibiotics revealed that B. humptydooensis and B. territorii clustered with B. pseudomallei strains from this study, and four of the five B. pseudomallei strains were included in the Ross et al.44 MMV compound study (see Supplemental Figure S1).

Table 2

Minimal inhibitory concentrations of MMV Pathogen Box compounds against B. humptydooensis and B. territorii strains compared with five B. pseudomallei strains

MIC (mg/L)*MIC (mg/L)
CompoundB. humptydooensis MSMB 43B. territorii MSMB 110B. pseudomallei K96243B. pseudomallei 576B. pseudomallei NCTC13178B. pseudomallei NCTC13179B. pseudomallei MX2013
Ceftazidime2–42–444463
Doxycycline0.40.3–112.532.52.5
Levofloxacin1–61–3410666
Rifampicin1313–264518182518
MMV6882714–84–861210812
MMV6759686–121> 0.72§> 0.72§> 0.72§> 0.72§> 0.72§

B. humptydooensis = Burkholderia humptydooensis; B. pseudomallei = Burkholderia pseudomallei; B. territorii = Burkholderia territorii; MIC = minimal inhibitory concentration; MMV = Medicines for Malaria Venture.

Minimal inhibitory concentration values for B. humptydooensis and B. territorii were determined from serial dilutions of the MMV Pathogen Box compounds, starting at 20 μM. Values were converted to mg/L. Data represent the MIC determined from three independent experiments.

Minimal inhibitory concentration values for B. pseudomallei were determined by Ross et al.44

Ceftazidime was not part of the MMV panel but was included as a positive control with potent activity toward B. pseudomallei.

Inhibitory activity toward B. pseudomallei was not detected at the tested concentration of 2 μM (0.72 mg/L).44

Newly identified compound MMV67968 showed activity toward B. humptydooensis and B. territorii with MICs (3–12 mg/L) that are less than MICs for rifampicin and only 2-fold higher than MICs for ceftazidime (2–6 mg/L), the “gold-standard” melioidosis therapy (see Table 2). Together, MMV compounds MMV688271 (Ross et al.44 study) and MMV67968 (this study) may provide useful information for developing new drugs with activity toward B. pseudomallei.

In summary, newly characterized nonpathogenic B. humptydooensis and B. territorii, and previously described B. thailandensis, recapitulate the antibiotic susceptibility of pathogenic B. pseudomallei strains, including clinical strains of Mexican, Thai, and Australian origin (MMV compound comparison).44 Therefore, each of these strains of nonpathogenic Burkholderia species has potential for use in high throughput in vitro screening of molecules for melioidosis therapy development.

The lower risk-group classification of the near-neighbor species allows expansion of melioidosis research into a wider landscape, where more laboratories have adequate facilities to perform the initial compound discovery. We are hopeful that inclusion of well-characterized and nonpathogenic model organisms in melioidosis research will accelerate the development of new treatment options for this neglected tropical disease.

Supplemental figure and tables

ACKNOWLEDGMENTS

We would like to thank Alysha Elliot at the Institute for Molecular Bioscience, the University of Queensland, for her critical review of this work; Quentin Kaas at the Institute for Molecular Bioscience, the University of Queensland, for his support with the principal component analysis; Medicines for Malaria Venture for supplying the Pathogen Box compounds; and Vanessa Rigas at the Menzies School of Health Research for her laboratory support.

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Author Notes

Address correspondence to Nicole Lawrence, Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Rd., Brisbane 4072, Australia, E-mail: n.lawrence@uq.edu.au or Sónia Troeira Henriques, Queensland University of Technology, School of Biomedical Sciences, Institute of Healthy and Biomedical Innovation, and Translational Research Institute, Brisbane, Queensland, 4102, Australia, E-mail: sonia.henriques@qut.edu.au.

Financial support: This work was supported by funding from the Australian government (A. S. A. Research Training Program Scholarship), the Australian National Health and Medical Research Council (J. R. W., M. M., and B. J. C. grant numbers 1046812, 1098337, and 1131932, respectively, and the HOT NORTH Initiative; D. J. C., S. T. H., and N. L. grant number 1084965), and the Australian Research Council (Laureate Fellowship to D. J. C. [FL150100146] and Future Fellowship to S. T. H. [FT150100398]).

Authors’ addresses: Anna S. Amiss, David J. Craik, and Nicole Lawrence, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia, E-mails: a.bundock@imb.uq.edu.au, d.craik@imb.uq.edu.au, and n.lawrence@uq.edu.au. Jessica R. Webb and Mark Mayo, Global and Tropical Health Division, Menzies School of Health Research, Darwin, Australia, E-mails: jessica.webb@menzies.edu.au and mark.mayo@menzies.edu.au. Bart J. Currie, Global and Tropical Health Division, Menzies School of Health Research, Darwin, Australia, and Northern Territory Medical Program, Department of Infectious Diseases, Royal Darwin Hospital, Darwin, Australia, E-mail: bart.currie@menzies.edu.au. Sónia Troeira Henriques, Queensland University of Technology, School of Biomedical Sciences, Institute of Healthy and Biomedical Innovation, and Translational Research Institute, Brisbane, Queensland, 4102, Australia, E-mail: sonia.henriques@qut.edu.au.

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