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    Survival rates of Caenorhabditis elegans. Approximately 30–40 age-matched C. elegans worms were coincubated with 106 CFU/mL Burkholderia pseudomallei vgh07, Burkholderia thailandensis E264, Burkholderia cenocepacia BC2, and Burkholderia multivorans NKI379 (A) or coincubated with B. pseudomallei strains vgh07, vgh45, vgh16W, and vgh16R (B) at 20°C. E. coli OP50 was used as normal food to feed C. elegans. The data are presented as the mean ± standard error of the mean. Letters a–d represent significant differences (P < 0.05, ANOVA with Tukey’s post hoc test). If two groups have a nonsignificant difference, same letter is shown.

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    In vitro association and invasion assay with peritoneal exudate cells (PECs). At MOI = 10:1 (Burkholderia pseudomallei-to-PECs), cells were grown in a 6-well plastic tray for 4 hours, and the PECs in the wells were harvested by centrifugation, washed with media (A) or washed with medium containing 400 μg/mL kanamycin to remove extracellular B. pseudomallei (B), and then disruption. After serial dilution, the number of PECs associated with or invaded by B. pseudomallei was determined by plate counting. Data are presented as the mean ± SD. Letters a–c represent significant differences (P < 0.05, ANOVA with Tukey’s post hoc test). If two groups have a nonsignificant difference, same letter is shown.

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    Burkholderia pseudomallei virulence in Caenorhabditis elegans and Dictyostelium discoideum models. The virulence of B. pseudomallei isolates (n = 33) was estimated based on C. elegans killing or resistance to D. discoideum predation. The survival rates (%) of C. elegans, shown on the y axis, were determined in liquid medium containing 30–40 age-matched worms and 106 CFUs/mL of individual B. pseudomallei isolates for 96 hours. The appearance of swarming zones or formation of fruiting bodies at different inocula (log CFU/mL) of individual B. pseudomallei isolates in the D. discoideum model is shown on the x axis. The red circle represents higher virulence against C. elegans and more resistance to predation by D. discoideum (strain NKE11); the pink circle represents higher virulence against C. elegans but more susceptibility to predation by D. discoideum (strain NKE23); the purple circle represents lower virulence against C. elegans but greater resistance to predation by D. discoideum (strain NKE10); and the blue circle represents lower virulence against C. elegans and greater susceptibility to predation by D. discoideum (strain NKE24). This figure appears in color at www.ajtmh.org.

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    Survival rates of mice with melioidosis. BALB/c mice (n = 10, each group) were intravenously infected with individual Burkholderia pseudomallei strains (100 CFU/mL) via the tail vein. The daily survival rates (%) are shown.

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    Bacterial loads in organs. Murine melioidosis was induced by Burkholderia pseudomallei strains NKE24, NKE23, NKE10, or B190 (100 CFU/mL) via intravenous injection. At the indicated times, the bacterial loads in the bone marrow, obtained by flushing liquid (1 mL) from the femur (A), or homogenized brains (0.4 g) (B) were determined by serial dilution and plate counting. At 30 day postinfection, bacterial loads in the spleen (0.02 g), liver (0.5 g), lungs (0.01 g), brain (0.4 g), and bone marrow (1 mL) of mice infected with strains NKE24 and B190 are shown (C). Data are presented as the mean ± standard error of the mean. Letters a–d represents significant differences (P < 0.05, ANOVA with Tukey’s post hoc test). If two groups have a nonsignificant difference, same letter is shown.

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    Inflamed cells or abscesses in the organs of mice with melioidosis. Representative abscesses (indicated by arrows) in the spleens (A, left, ×10; right, ×100) and cellular debris (indicated by arrows; B, left, ×100) or regenerated cells (indicated by arrows; B, right, ×400) in the livers were found. In the lung, neutrophil infiltration (indicated by arrows; C, left, ×400) and pulmonary emboli (indicated by arrows; C, right, ×100) are shown. Microabscess (indicated by arrows) appeared in the lumbar vertebrate (D, left, ×200) and femur (D, right, ×40). The infiltration of inflamed cells (indicated by arrows; E, ×100) in the submandibular gland and degenerated neutrophils (indicated by arrows; F, ×400) in the parotid gland as well as the degenerated inflamed cells in the nerves (indicated by arrows; G, ×100), and meningeal suppuration (H, bottom left, ×40) and a number of necrotic debris (indicated by arrows; H, ×100) are shown.

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Distinct Pathogenic Patterns of Burkholderia pseudomallei Isolates Selected from Caenorhabditis elegans and Dictyostelium discoideum Models

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  • 1 Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung, Taiwan;
  • 2 Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan;
  • 3 Medical Research Department, General Clinical Research Center, Taipei Veterans General Hospital, Taipei, Taiwan;
  • 4 School of Medicine, Institute of Public Health, National Yang-Ming University, Taipei, Taiwan;
  • 5 Department of Internal Medicine, National Yang-Ming University, Taipei, Taiwan

Burkholderia pseudomallei is a selective agent that causes septic melioidosis and exhibits a broad range of lethal doses in animals. Host cellular virulence and phagocytic resistance are pathologic keys of B. pseudomallei. We first proposed Caenorhabditis elegans as the host cellular virulence model to mimic bacterial virulence against mammals and second established the resistance of B. pseudomallei to predation by Dictyostelium discoideum as the phagocytosis model. The saprophytic sepsis–causing Burkholderia sp. (B. pseudomallei, Burkholderia thailandensis, Burkholderia cenocepacia, and Burkholderia multivorans) exhibited different virulence patterns in both simple models, but B. pseudomallei was the most toxic. Using both models, attenuated isolates of B. pseudomallei were selected from a transposon-mutant library and a panel of environmental isolates and reconfirmed by in vitro mouse peritoneal exudate cell association and invasion assays. The distinct pathological patterns of melioidosis were inducted by different selected B. pseudomallei isolates. Fatal melioidosis was induced by the isolates with high virulence in both simple models within 4–5 day, whereas the low-virulence isolates resulted in prolonged survival greater than 30 day. Infection with the isolates having high resistance to D. discoideum predation but a low C. elegans killing effect led to 83% of mice with neurologic melioidosis. By contrast, infection with the isolates having low resistance to D. discoideum predation but high C. elegans killing effect led to 20% cases with inflammation in the salivary glands. Our results indicated that individual B. pseudomallei isolates selected from simple biological models contribute differently to disease progression and/or tissue tropism.

INTRODUCTION

As a soil-borne pathogen, Burkholderia pseudomallei is the causative agent of melioidosis and is endemic to tropical regions such as Southeast Asia and northern Australia.1 The transmission modes of melioidosis include subcutaneous inoculation, inhalation, and, to a lesser extent, ingestion.2,3 The symptoms of melioidosis patients are variable, including localized cutaneous suppuration, multiple organ abscesses, sepsis, and neurological disorders, but pulmonary melioidosis with or without septicemia is commonly observed.4 Severe progression of melioidosis is dependent on inoculum size, bacterial virulence, routes of host invasion, and the host immune response.5,6 Particularly in animal models, virulent B. pseudomallei strains isolated from different sources exhibit broad lethal doses, ranging from < 10 to 107 colony-forming units (CFUs).5,7 Different virulence patterns could exist in B. pseudomallei isolates.

However, animal studies of B. pseudomallei are usually constrained by rigorous regulations in most countries because B. pseudomallei could potentially be used as a biological weapon.8 Alternatively, the simple, consistent, standardized, and quality-controlled model organisms Caenorhabditis elegans and Dictyostelium discoideum have been developed as surrogates of the mammalian host to estimate the virulence of B. pseudomallei.9,10 Bacterial attachment to host cells is the first step in the establishment of B. pseudomallei infection.11 The soil nematode C. elegans is a reasonable model because this organism is susceptible to the attachment and proliferation of B. pseudomallei in the intestinal lumen.12,13 As an intracellular pathogen, B. pseudomallei–loaded cells, similar to the Trojan horse, can be disseminated throughout the body via a hematogenous route.1416 Dictyostelium discoideum, a unicellular protozoan, digests B. pseudomallei via well-characterized phagosomal and endolysosomal mechanisms similar to those associated with cytoskeletal organization, membrane trafficking, and endocytic events in mammalian macrophages.10 Thus, attenuation of persistence in D. discoideum, a surrogate of mammalian phagocytic cells, could result in reduction of B. pseudomallei dissemination inside the host body.

In this study, B. pseudomallei vgh07, Burkholderia thailandensis E264, Burkholderia cenocepacia BC2, and Burkholderia multivorans NKI379 are used for comparative interspecies analysis of Burkholderia bacteria because these species are soil-borne pathogens that opportunistically cause pulmonary melioidosis or cepacia syndrome.1719 Burkholderia pseudomallei vgh07 and B. cenocepacia BC2 were isolated from septic patients, whereas B. thailandensis E264 and B. multivorans NKI379 were isolated from rice field soil. In particular, B. thailandensis E264 is genetically closely related to B. pseudomallei strains but is a weakly virulent relative.20 Burkholderia multivorans NKI379 was unable to colonize a mouse model by intravenous infection.21 However, Burkholderia bacteria usually had different degrees of C. elegans killing or of resistance to predation by D. discoideum in a co-cultivated system.10,2226 In an attempt to identify attenuated isolates from transposon-derived mutants and screen distinct virulence patterns from environmental isolates of B. pseudomallei, likely exhibiting different abilities of bacterial attachment or anti-phagocytic activity, both C. elegans and D. discoideum models were used as surrogates of mammals in this study. Furthermore, different pathological patterns associated with melioidosis were characterized in animal models.

MATERIALS AND METHODS

Ethics statement.

The Bagg and Albino/c (BALB/c) mouse experiments performed in this study were approved by the Institutional Review Board (Institutional Animal Care and Use Committee [IACUC]/Ethics Committee, approval number, 10301 and 10403; National Kaohsiung Normal University [NKNU], Taiwan) and performed in accordance with the Animal Protection Act (Taiwan). All experiments manipulated for viable B. pseudomallei were performed in a biosafety level III laboratory at Kaohsiung Veterans General Hospital, Taiwan.

Organisms and animals.

All B. pseudomallei isolates used in this study are listed in Table 1. To conduct experiments on environmental isolates, a total of 33 B. pseudomallei isolates (sequencing types (STs) obtained by multilocus sequence typing; ST58, n = 10; ST704, n = 3; ST834, n = 4; ST1001, n = 10; ST1115, n = 2; and ST1354, n = 4; shown in Supplemental Table 1) were isolated from soil or water in the Zoynan Region, southern Taiwan.3

Table 1

Bacterial strains and plasmids used in this study

Organisms or plasmidRelevant propertiesReference
Organisms
 Caenorhabditis elegans N2Wild type; nematode; obtained from National Cheng Kung University27
 Dictyostelium discoideum AX2Wild type; slime mode; obtained from Dicty Stock CenterThis study
Bacteria
 Burkholderia thailandensis E264Isolated from rice paddy soil; obtained from ATCCATCC700388
 Burkholderia cenocepacia BC2Isolated from patients with sepsis21
 Burkholderia multivorans NKI379Isolated from rice paddy soilBioSample: SAMN04008114
 Burkholderia pseudomallei
  vgh07Wild type; isolated from bacteremia patients (ST58); high virulenceBioSample: SAMN03372474
  vgh45Wild type; isolated from bacteremia patients (ST1001); low virulence7
  vgh16WIsogenic strain; derived from vgh16; relatively low virulence compared with vgh16R vgh16RBioSample: SAMN04009760
  vgh16RIsogenic strain; derived from vgh16; relatively high virulence compared with vgh16WVgh16WBioSample: SAMN04009759
  B190Attenuated strain; selected from a Tn5 transposon libraryThis study
 E. coli DH5αF– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dLacZΔM15Δ(lacZYA-argF)U169, and hsdR17 (rK–mK+), λ–; used for cloning14
  λpirsup E44, ΔlacU169 (ΦlacZΔM15), recA1, endA1, hsdR17, thi-1, gyrA96, relA1, and λpir; used for maintenance of plasmids containing R6K ori14
  OP50Used for maintenance of C. elegans strains27
 Klebsiella aerogenesUsed for maintenance of D. discoideum strains; obtained from Dicty Stock CenterThis study
Plasmids
pKNOCKMobilized suicide vector, cmr, R6K ori14
pRK2013Helper plasmid for triparental conjugation14

ST = sequencing type.

Information regarding the simple biological models C. elegans N2 and D. discoideum AX2, their reference strains Escherichia coli OP50 and Klebsiella aerogenes, respectively, and Burkholderia sp. (B. thailandensis, B. cenocepacia, and B. multivorans) is shown in Table 1. All of the bacteria were stored in Luria–Bertani (LB) broth at −80°C. Before the experiments, the bacteria were revived in LB broth at 37°C for 16 hours. Caenorhabditis elegans N2 was maintained in static nematode growth medium containing monoxenic E. coli OP50 at 20°C. Thawing, culturing, cleaning for egg preparation, and synchronization of young worms were performed according to standard protocols.27 About 5 × 104 cells/mL of D. discoideum AX2 was inoculated in HL5 axenic medium (5 g of proteose peptone, 5 g of thiotone E peptone, 10 g of glucose, 5 g of yeast extract, 0.35 g of Na2HPO4, 0.35 g of KH2PO4, and 0.05 g of dihydrostreptomycin sulfate per liter) and shaken at 22°C, 180 rpm for 2–3 day until an exponential growth phase (2–6 × 106 cells/mL) was reached. Unicellular densities were monitored by microscopic examination at a ×200 magnification using a hemocytometer. Preparation of unicellular protozoans in log phase was performed according to standard protocols.28

All mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan), and the experiments were performed under specific pathogen–free and air flow–controlled conditions.

Caenorhabditis elegans survival assay.

To record the movement, the C. elegans survival assay was performed in a clear liquid medium (22 g of KH2PO4, 7 g of K2HPO4, and 1% (w/v) glucose per liter). Briefly, overnight cultures of the individual isolates were adjusted to 107 CFUs/mL of bacterial suspension using the liquid media. The 20-μL bacterial suspension was added to 3 mL of 40–50 age-matched worms at young adult stage in the liquid media. The total numbers of C. elegans in the media were counted using automatic video recording (iCATCH, DVR-413DH-J; Panasonic, Taipei, Taiwan). Nematodes showing rapid undulating movement were recognized as alive, whereas as dead when they did not move for more than 3 seconds in the video recording. Survival rates (%) were monitored daily. Assays were performed at 20°C, and E. coli OP50 was used as a normal food control.

Resistance to predation by D. discoideum.

Klebsiella aerogenes (2 × 106 CFU/mL) and the tested bacteria at final concentrations ranging from 100 to 106 CFU/mL were mixed and then plated on SM-Agar (10 g glucose, 10 g proteose peptone, 1 g yeast extract, 0.5 g MgSO4, 1.9 g KH2PO4, 0.6 g K2HPO4, and 20 g bacto agar per liter) plates. As controls, K. aerogenes ranging from 103 to 106 CFU/mL were plated on SM-Agar. Log-phase D. discoideum cells were adjusted to 2 × 106 cells/mL by counting using a hemocytometer. One drop (10 μL) of D. discoideum cells was deposited 10 mm from the center of each plate. The plates were incubated at 22°C, and the formation of plaques or fruiting bodies in the bacterial lawns was recorded after 120 hours.

In vitro association and invasion assay.

Mouse peritoneal exudate cells (PECs) were isolated by peritoneal lavage 4 day after intraperitoneal injection of 2 mL of sterile 3% Brewer Thioglycollate medium (Difco Laboratories, Detroit, MI). The PECs were washed and suspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal calf serum (FCS) and penicillin and streptomycin (100 μg/mL) at a final concentration of 105 cells/mL. Both in vitro PEC association and invasion assays were performed at an multiplicity of cellular infection (MOI) = 10:1 (105 CFU of B. pseudomallei to 105 cells of PECs) in a 6-well plastic tray. After 4 hours, for the in vitro association assay, total PECs in the wells were harvested by centrifugation (180 × g), washed with RPMI 1640 medium, and disrupted with 100 μL of 1% Triton X-100. After serial dilution, the numbers of B. pseudomallei were determined by plate counting. For the in vitro invasion assay, the PECs were washed with RPMI 1640 medium containing 400 μg/mL kanamycin to remove extracellular B. pseudomallei. After three washes, the infected PECs were disrupted, and the number of intracellular bacteria (log cells/105 PEC) was determined by plate counting.

Construction of a Tn5 transposon-mutant library.

The Tn5-mutant library was generated by a triparental mating system. For conjugation, E. coli DH5α pir pKNOCK (miniTn5, pfliC-lacZ-cat; Cmr, Aps; 109 CFU) was used as a donor strain, E. coli pRK2013 (109 CFU; Cms, Aps) was used as a helper strain and B. pseudomallei vgh07 was the recipient (109 CFU; Cms, Apr); the three strains were mixed and filtered onto cellulose paper (< 0.45 μm). Burkholderia pseudomallei mutants were selected for chloramphenicol resistance and screened for blue colonies after hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside; Sigma Co., St. Louis, MO).

A three-step process was used for mutant selection. First, mutants exhibiting moist, smooth colonies on Ashdown’s medium after a 7-day incubation were collected. Then, these mutants were seeded in minimal medium (4 g of glucose, 1 g of NH4Cl, 6 g of Na2HPO4, 3 g of KH2PO4, and 0.5 g of NaCl per liter). After a 3-day incubation, thriving mutants were collected from the medium to exclude auxotrophic mutants. Then, the selected mutants were evaluated in C. elegans and D. discoideum models (see previous paragraphs). Eventually, the attenuated mutant B190 was selected from this study because this mutant exhibited > 50% survival rates in C. elegans, and in the D. discoideum model, evident digestion (formation of plaque or fruiting bodies) appeared with 104 CFUs of inoculum.

Survival rates of mice with melioidosis and bacterial loads in organs.

Eight-week-old female BALB/c mice (n = 10) were intravenously injected with B. pseudomallei (50 μL; 100 CFU/mouse) via the tail vein. The survival rates (%) were recorded daily.

Mice with melioidosis (n = 6) were sacrificed at 1, 10, 12, 15, and 20 day postinfection. The solid organs (spleen, 0.02 g; liver, 0.5 g; lung, 0.01 g; and brain, 0.4 g) were excised and homogenized in 500 mL of 2% FCS-phosphate-buffered saline (PBS) using tissue grinders (Thermo Fisher Scientific Inc., Fremont, CA). The bone marrow cells were aseptically flushed from the femur using 1 mL of 2% FCS-PBS. The total number of bacteria in the bone marrow (CFU/mL) or organs (CFU/g) was determined using serial dilutions and the plate count method. The limits of detection were 20, 50, 50, and 3 CFUs/g for the liver, spleen, lung, and brain, respectively, and 10 CFUs/mL for bone marrow.

Histological examination.

To observe the inflamed cells or microabscesses in the organs, mice with melioidosis were sacrificed at 1 day postinfection for the vgh07 (trials, n = 8) and NKE11 (trials, n = 6) infection groups and at 10 day postinfection for the NKE24 (trials, n = 7), NKE23 (trials, n = 10), NKE10 (trials, n = 6), and B190 (trials, n = 10) infection groups. The skull, vertebrae, and limbs were excised, fixed in 4% formaldehyde, and decalcified with 10% trichloroacetic acid. Other solid organs, such as the heart, lung, spleen, liver, bladder, and kidney, were fixed in 4% formaldehyde. All tissues were processed for embedding in paraffin wax using standard techniques.14 Sectioning was performed at appropriate sites for the parietal lobe and salivary gland from the whole skull, bone, and skeletal muscles from the limbs, nerves from the vertebrae, and bone marrow from the vertebrae and limbs using a customized cutting box. Neutrophil or lymphocyte infiltration, necrosis, and abscesses were observed using hematoxylin and eosin staining.

Statistical analysis.

Data are presented as the mean ± standard error of the mean or mean ± SD. Variable comparisons were analyzed using analysis of variance (ANOVA) and Tukey’s honest significant difference (HSD) test. Differences in the presence or absence of inflammatory foci were examined by Fisher’s exact test (one-tailed). Significance was set at a level of P < 0.05.

Accession numbers.

The nucleotide sequences obtained in this study are available at the National Center for Biotechnology Information under the following GenBank numbers: CP010973.1 (chromosome 1) and CP010974.1 (chromosome 2).

RESULTS

Caenorhabditis elegans survival.

According to our results, C. elegans was most susceptible to B. pseudomallei among the four Burkholderia sp. tested. After 96 hours, the survival rates of C. elegans were 7.9 ± 1.8% for the B. pseudomallei vgh07 group, 50.8 ± 3.4% for the B. thailandensis E264 group, 70.9 ± 3.4% for the B. cenocepacia BC2 group, and 54.7 ± 4.6% for the B. multivorans NKI379 group (Figure 1A). Similar survival rates of C. elegans were observed for the B. thailandensis E264 and B. multivorans NKI379 groups after 96 hours. Notably, the environmental strain B. thailandensis E264 rarely causes human disease, but the genomic background of this strain is similar to that of B. pseudomallei strains.29 Burkholderia cenocepacia BC2 was isolated from patients, whereas B. multivorans was isolated from cropped soil.21,30

Figure 1.
Figure 1.

Survival rates of Caenorhabditis elegans. Approximately 30–40 age-matched C. elegans worms were coincubated with 106 CFU/mL Burkholderia pseudomallei vgh07, Burkholderia thailandensis E264, Burkholderia cenocepacia BC2, and Burkholderia multivorans NKI379 (A) or coincubated with B. pseudomallei strains vgh07, vgh45, vgh16W, and vgh16R (B) at 20°C. E. coli OP50 was used as normal food to feed C. elegans. The data are presented as the mean ± standard error of the mean. Letters a–d represent significant differences (P < 0.05, ANOVA with Tukey’s post hoc test). If two groups have a nonsignificant difference, same letter is shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 4; 10.4269/ajtmh.19-0052

To test the differential virulence among B. pseudomallei strains, four representative B. pseudomallei strains were coincubated with C. elegans. Of these strains, B. pseudomallei vgh16R (a smooth colony morphovar) and vgh16W (a wrinkled colony morphovar), which belonged to an isogenic group, were spontaneously derived from a parent strain vgh16 (a wrinkled colony morphovar) when subcultured in Ashdown’s media. Previously, we demonstrated that the virulence of isogenic strain vgh16W was significantly higher than that of isogenic strain vgh16R in a BALB/c mouse mode.17,31 Our results indicated a rapid decrease in C. elegans survival rates in the B. pseudomallei vgh07 and vgh16W groups but a slow decrease in the B. pseudomallei vgh16R and vgh45 groups (Figure 1B).

Resistance to predation by D. discoideum.

In the D. discoideum model, formation of plaque or fruiting bodies was indicative of digestion of the bacteria by D. discoideum. The strain K. aerogenes is known to be susceptible to predation by D. discoideum. By 72 hours, plaque was formed in the K. aerogenes lawn with an inoculum of 103–106 CFUs, and the bacteria were subjected to complete predation and replaced by D. discoideum fruiting bodies after 120 hours. In a comparison of the four Burkholderia species, plaques or fruiting bodies were observed in the B. pseudomallei vgh07 lawn at an inoculum of 102 CFUs, the B. multivorans NKI379 lawn at an inoculum of 103 CFU, and the B. thailandensis E264, and B. cenocepacia BC2 lawns at an inoculum of 104 CFUs (Table 2). Burkholderia pseudomallei vgh07 was the most resistant to predation by D. discoideum. The level of resistance to D. discoideum predation was not related to the C. elegans killing effects in the B. thailandensis and B. cenocepacia groups.

Table 2

Dictyostelium discoideum predation assay

StrainsPresence (P) or absence (A) of plaques or fruiting bodies* at the indicated inoculum concentration (CFU/mL)
101102103104105106
Klebsiella aerogenesPPPP
Burkholderia cenocepacia BC2PPPPAA
Burkholderia multivorans NKI379PPPAAA
Burkholderia thailandensis E264PPPPAA
Burkholderia pseudomallei vgh07PPAAAA
B. pseudomallei vgh45PPAAAA
B. pseudomallei 16WPPAAAA
B. pseudomallei 16RPPPAAA
B. pseudomallei B190PPPPAA

CFU = colony-forming unit.

* Observed by 120 hours.

Regarding the difference in the resistance to predation of B. pseudomallei, the strains vgh07 and vgh45 and both the isogenic strains vgh16W and vgh16R at an inoculum of 101–104 CFUs were added into the D. discoideum predation model. In the case of strain vgh16R, the formation of plaques or fruiting bodies was observed at an inoculum of 103 CFU. However, both signs of resistant to predation were observed at an inoculum of 102 CFUs of strains vgh07, vgh45, and vgh16W (Table 2). Interestingly, B. pseudomallei vgh45 was resistant to predation by D. discoideum but exhibited the least virulence against C. elegans among the B. pseudomallei strains. The intergenus patterns of Burkholderia sp. and interspecies patterns of B. pseudomallei strains appeared to be different in terms of virulence in C. elegans and D. discoideum models.

Applications in the discovery of attenuated mutants.

To select an attenuated mutant by using both the simple biological models, a Tn5 library was constructed by triparental conjugation. This library contained 2,470 smooth-colony strains and 7,260 wrinkled-colony strains. Because 1) we previously found that the virulence in BALB/c models of B. pseudomallei with smooth colonies was usually attenuated compared with that of isolates with wrinkled colonies7,32 and 2) auxotrophic mutants needed to be excluded from the library, approximately 1,290 mutants that thrived in minimal media were selected from the 2,470 smooth-colony mutants. To rapidly screen the attenuated mutants, the survival of C. elegans cocultivated with individual mutants in a 96-well plate was automatically recorded, and worm motility was qualitatively evaluated by video recording. A total of 26 individual mutants exhibited > 50% survival rates of C. elegans after 96 hours (Supplemental Figure 1A). Furthermore, 15.4% (4/26) of these mutants simultaneously exhibited high susceptibility to predation by D. discoideum (formation of plaques or fruiting bodies at an inoculum of 104 CFUs). The representative mutant B. pseudomallei B190 was selected for further study. According to the inverse polymerase chain reaction (PCR) and resultant sequences, the insertion sites were proposed to be in the glucosyl transferase gene (Supplemental Figure 1B).

To confirm that the results derived from the simple biological models reflected virulence attenuation in mammalian cells, in vitro PEC association and invasion assays were performed with the B. pseudomallei strains. High numbers of B. pseudomallei vgh07 and vgh16W were observed in both the PEC association and invasion assays (Figure 2). Both B. pseudomallei B190 and vgh16R exhibited attenuation in the in vitro PEC association and invasion assays compared with the B. pseudomallei vgh07 and vgh16W groups (cell association assay, F4,29 = 76.30, P < 0.05; cell invasion assay, F4,29 = 293.66, P < 0.05; ANOVA). Burkholderia pseudomallei B190 was the most attenuated strain (compared with all other strains, P < 0.05, Tukey’s HSD test). Different patterns of virulence were observed in the B. pseudomallei vgh45 group, which exhibited low numbers in the association assay, but the pattern observed in the in vitro PEC invasion assay was identical to that observed for the B. pseudomallei vgh07 and vgh16W groups.

Figure 2.
Figure 2.

In vitro association and invasion assay with peritoneal exudate cells (PECs). At MOI = 10:1 (Burkholderia pseudomallei-to-PECs), cells were grown in a 6-well plastic tray for 4 hours, and the PECs in the wells were harvested by centrifugation, washed with media (A) or washed with medium containing 400 μg/mL kanamycin to remove extracellular B. pseudomallei (B), and then disruption. After serial dilution, the number of PECs associated with or invaded by B. pseudomallei was determined by plate counting. Data are presented as the mean ± SD. Letters a–c represent significant differences (P < 0.05, ANOVA with Tukey’s post hoc test). If two groups have a nonsignificant difference, same letter is shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 4; 10.4269/ajtmh.19-0052

Evaluation of B. pseudomallei from environmental isolates with different virulence using biological models.

To test the different virulence of B. pseudomallei isolates, a panel of environmental B. pseudomallei isolates (ST58, n = 10; ST704, n = 3; ST834, n = 4; ST1001, n = 10; ST1115, n = 2; ST1354, n = 4) were used to conduct virulence assays in both biological models (Supplemental Table 1). On average, 17.1 ± 7.3% survival was observed in C. elegans coincubated with individual B. pseudomallei isolates. The formation of plaques and fruiting bodies of D. discoideum on B. pseudomallei lawns was observed with an inoculum of log 2.5 ± 0.7 CFU/mL. Four of the B. pseudomallei isolates exhibited outlier patterns in the C. elegans or D. discoideum models (Figure 3). For example, B. pseudomallei NKE11 exhibited high virulence against C. elegans and resistance to predation by D. discoideum; B. pseudomallei NKE23 exhibited high virulence against C. elegans but was susceptible to predation by D. discoideum; B. pseudomallei NKE10 exhibited low virulence against C. elegans but was resistant to predation by D. discoideum; and B. pseudomallei NKE24 exhibited low virulence against C. elegans and was susceptible to predation by D. discoideum. The results for neither the C. elegans (Supplemental Figure 2A) nor D. discoideum (Supplemental Figure 2B) model were related to the STs.

Figure 3.
Figure 3.

Burkholderia pseudomallei virulence in Caenorhabditis elegans and Dictyostelium discoideum models. The virulence of B. pseudomallei isolates (n = 33) was estimated based on C. elegans killing or resistance to D. discoideum predation. The survival rates (%) of C. elegans, shown on the y axis, were determined in liquid medium containing 30–40 age-matched worms and 106 CFUs/mL of individual B. pseudomallei isolates for 96 hours. The appearance of swarming zones or formation of fruiting bodies at different inocula (log CFU/mL) of individual B. pseudomallei isolates in the D. discoideum model is shown on the x axis. The red circle represents higher virulence against C. elegans and more resistance to predation by D. discoideum (strain NKE11); the pink circle represents higher virulence against C. elegans but more susceptibility to predation by D. discoideum (strain NKE23); the purple circle represents lower virulence against C. elegans but greater resistance to predation by D. discoideum (strain NKE10); and the blue circle represents lower virulence against C. elegans and greater susceptibility to predation by D. discoideum (strain NKE24). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 4; 10.4269/ajtmh.19-0052

Induction of melioidosis in mice.

We proposed that the B. pseudomallei isolates selected from the C. elegans and D. discoideum models would potentially exhibit different pathogenic patterns in animal models. Thus, melioidosis was induced in mice via intravenous injection of the representative strains vgh07, B190, NKE11, NKE10, NKE23, and NKE24. The survival rates were recorded daily. All of the mice infected with the strains vgh07 and NKE11, which are likely high-virulence strains, died within 4 to 5 d. However, the survival rates of the mice infected with strains NKE23 and NKE10 were prolonged to 15-20 d. Regarding the attenuated strains B190 and NKE24, ≥ 50% survival was recorded over a 30-d infection period (Figure 4).

Figure 4.
Figure 4.

Survival rates of mice with melioidosis. BALB/c mice (n = 10, each group) were intravenously infected with individual Burkholderia pseudomallei strains (100 CFU/mL) via the tail vein. The daily survival rates (%) are shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 4; 10.4269/ajtmh.19-0052

All of the mice with melioidosis exhibited B. pseudomallei loads in the liver and spleen after 1 d (Supplemental Table 2). However, bacterial loads in the bone marrow or brain were detected at different times. For example, the mice infected with B. pseudomallei NKE10 exhibited bacterial loads in the bone marrow (Figure 5A) and brain (Figure 5B) at 10 day postinfection. For both strains NKE23 and NKE24, bacterial loads were present in the bone marrow or brain at 12 day postinfection. Nevertheless, the bacterial loads in the spleen and liver were detected in the mice infected with the attenuated strain B. pseudomallei B190 and strain NKE24 at 20 day postinfection (Figure 5C).

Figure 5.
Figure 5.

Bacterial loads in organs. Murine melioidosis was induced by Burkholderia pseudomallei strains NKE24, NKE23, NKE10, or B190 (100 CFU/mL) via intravenous injection. At the indicated times, the bacterial loads in the bone marrow, obtained by flushing liquid (1 mL) from the femur (A), or homogenized brains (0.4 g) (B) were determined by serial dilution and plate counting. At 30 day postinfection, bacterial loads in the spleen (0.02 g), liver (0.5 g), lungs (0.01 g), brain (0.4 g), and bone marrow (1 mL) of mice infected with strains NKE24 and B190 are shown (C). Data are presented as the mean ± standard error of the mean. Letters a–d represents significant differences (P < 0.05, ANOVA with Tukey’s post hoc test). If two groups have a nonsignificant difference, same letter is shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 4; 10.4269/ajtmh.19-0052

By histological examination, the abscesses were found in multiple organs, including spleen, liver, lung, bone marrow, nerves, and brain. Splenic and hepatic abscesses were common features in all the mice with melioidosis. The spleens usually became enlarged and exhibited multiple microabscesses (Figure 6A, left). Large amounts of cellular debris were observed in the center of the microabscess (Figure 6A, right). Necrotic hepatocytes or cellular debris and degenerated neutrophils were observed in hepatic microabscess (Figure 6B, left). Occasionally, the necrotic hepatocytes or cellular debris were replaced with regenerated cells by infiltration (Figure 6B, right). Neutrophils infiltrated and diffused into the alveoli (Figure 6C, left) or became pulmonary emboli (Figure 6C, right). Many but not all mice with melioidosis exhibited microabscess in the bone marrow of the lumbar vertebrae (Figure 6D, left) or femur (Figure 6D, right). In the cases of salivary glands, the infiltration of many inflamed cells in submandibular gland (Figure 6E) or degenerated neutrophil in the parotid gland (Figure 6F) was observed. In cases of B. pseudomallei NKE10 infection, development of microabscess in nerve (Figure 6G) and meningeal suppuration (Figure 6H) were observed. A number of necrotic debris was present in the foci (Figure 6H).

Figure 6.
Figure 6.

Inflamed cells or abscesses in the organs of mice with melioidosis. Representative abscesses (indicated by arrows) in the spleens (A, left, ×10; right, ×100) and cellular debris (indicated by arrows; B, left, ×100) or regenerated cells (indicated by arrows; B, right, ×400) in the livers were found. In the lung, neutrophil infiltration (indicated by arrows; C, left, ×400) and pulmonary emboli (indicated by arrows; C, right, ×100) are shown. Microabscess (indicated by arrows) appeared in the lumbar vertebrate (D, left, ×200) and femur (D, right, ×40). The infiltration of inflamed cells (indicated by arrows; E, ×100) in the submandibular gland and degenerated neutrophils (indicated by arrows; F, ×400) in the parotid gland as well as the degenerated inflamed cells in the nerves (indicated by arrows; G, ×100), and meningeal suppuration (H, bottom left, ×40) and a number of necrotic debris (indicated by arrows; H, ×100) are shown.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 4; 10.4269/ajtmh.19-0052

In summary, hepatic and splenic abscesses were commonly observed in mice with melioidosis (100% of melioidosis mice). In this study, signs of inflammation were never observed in the bladder, kidney, and heart. Approximately > 80% (vgh07 and NKE11 groups), 33–43% (NKE24, NKE23, and NKE10 groups), or 10% (B190 group) of mice with melioidosis showed pulmonary abscesses and/or neutrophil infiltration. However, inflammation in salivary gland (20%) was found in the B. pseudomallei NKE23–infected groups, and a large number of mice (83%) exhibited neurologic melioidosis in the NKE10-infected groups (Table 3). Burkholderia pseudomallei B190 infection was particularly attenuated. Except for the liver and spleen, 0–10% microabscess formation was observed in the tested organs. Overall, different pathogenic patterns in terms of survival rates, bacterial loads in organs, and microabscess formation were observed in animals infected with different B. pseudomallei isolates selected from distinct virulence in C. elegans and D. discoideum models.

Table 3

Pathogenic patterns in the BALB/c mouse model

Percentages of histologically inflammatory foci for the melioidosis mice (n = number) induced by the strains
vgh07 (n = 8)NKE11 (n = 6)NKE24 (n = 7)NKE23 (n = 10)NKE10 (n = 6)B190 (n = 10)
Foci appeared in
 Spleen100%100%100%100%100%100%
 Liver100%100%100%100%100%100%
 Lung88%a83%a43%ab40%ab33%ab10%b
 Bone marrow0%17%ab57%a70%a83%a10%b
 Salivary gland0%0%0%20%17%0%
 Nerves0%0%29%40%50%10%
 Brain or meninges0%0%14%b10%b83%a0%

Letters a–b represent that significant differences were existed between two groups. If two groups have a nonsignificant difference, same letter is shown (P < 0.05, Fisher’s exact test (one-tailed); statistical test was not performed for which data were 0%).

DISCUSSION

Using the simple biological models C. elegans and D. discoideum, attenuated isolates of B. pseudomallei were rapidly isolated from a transposon-mutant library or a panel of environmental isolates in this study. The pathogenic patterns varied in the BALB/c melioidosis model induced by individual B. pseudomallei isolates, which were selected based on C. elegans killing and resistance to D. discoideum predation. Highly virulent isolates likely caused acute melioidosis and exhibited high bacterial loads in the spleen and liver, eventually leading to death within 5 day. The manifestation of melioidosis for the mice infected with low-virulence isolates was varied, including different bacterial loads in organs and distribution of microabscesses in organs, eventually leading to variable survival rates. The different pathological patterns of melioidosis still threatened life, even caused infection with an attenuated isolate, as both the liver and spleen were found to become reservoirs of B. pseudomallei during the melioidosis.

Burkholderia sp., which are soil-borne pathogens, are the causative agents of septic melioidosis (B. pseudomallei or, to a lesser extent, B. thailandensis) and cepacia syndrome (B. cenocepacia or B. multivorans).3335 In terms of evolution, the virulence of soil-borne pathogens is enhanced by long-term adaptive selection in environmental hosts (bodies or cadaver).36 For example, at least the quorum sensing system, which is an intercellular communication pathway that responds to environmental-to-host changes, was conserved among the Burkholderia sp. This conserved system controls the production of many virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation.37 Thus, it is not surprising that B. pseudomallei, B. thailandensis, B. cenocepacia, and B. multivorans exhibited C. elegans killing in this study.22,24,38,39 Caenorhabditis elegans is reported to be highly susceptible to toxins (malleilactone or endotoxin) that are secreted by B. pseudomallei and to bacterial attachment and proliferation on the intestinal epithelium.12,40 The high rate of C. elegans killing exhibited by B. pseudomallei indicates that melioidosis is the most severe mammalian disease caused by the four sepsis-causing bacteria. Moreover, Burkholderia sp. (B. pseudomallei, B. thailandensis, B. cenocepacia, and B. multivorans) are capable of persisting in phagocytic cells for long periods.41,42 Burkholderia pseudomallei– or B. thailandensis–loaded cells can escape the phagolysosome, replicate in the cytosol and form multinucleated giant cells that contribute to cell-to-cell dissemination in vitro.43 CD11b+Ly6C+ monocytes harboring B. pseudomallei lead to high risks of whole-body bacterial dissemination in animal models.14,15,44 The bacterial genes required for resistance to predation by D. discoideum are similar to those expressed in mammalian phagocytic cells.10,45 Our results indicated that B. pseudomallei exhibited the strongest resistance to predation by D. discoideum and that, in humans, B. pseudomallei–induced melioidosis is more severe than other Burkholderia sp.–induced diseases.

By using C. elegans and D. discoideum models, we selected the attenuated B. pseudomallei B190 (proposed to be an insertion mutant of the glycosyl transferase gene that is potentially involved in capsular polysaccharide synthesis) from the transposon mutant library. Capsular polysaccharides in B. pseudomallei isolates are a known virulent factor determinant involved in survival in vivo, multiple cytokine responses, neutrophil infiltration, and acute inflammation in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa.46 In this study, among animals infected with B. pseudomallei B190, the survival (> 60%) of mice with melioidosis was prolonged to more than than 30 d. By contrast, strains vgh07 and NKE11 exhibited high rates of C. elegans killing and high resistance to D. discoideum predation, resulting in deaths of all mice with melioidosis within 4–5 day. However, B. pseudomallei has multiple virulence factors, including those involved in cell entry, vesicle formation, anti-phagocytosis activity, phagosome escape, multiplication, and formation of multinucleated giant cells.46 Obviously, neither biological model can fully reflect all the processes associated with B. pseudomallei invasion of mammalian cells. However, notably, infection with strain NKE10, with high resistance to D. discoideum predation but low virulence against C. elegans, caused more cases (83%) with neurologic melioidosis than infection with other B. pseudomallei isolates. Intracellular survival and the formation of multinucleated giant cells in mammals have been reported to contribute to the pathogenesis observed in B. pseudomallei infection.43 Persistence of B. pseudomallei in phagocytic cells is associated with a particularly high risk for the development of neurologic melioidosis.14 Burkholderia pseudomallei vgh45 exhibited low rates of C. elegans killing but high resistance to D. discoideum predation. Accordingly, this strain exhibited low degrees of in vitro PEC association, likely leading to loss of the ability of attachment to C. elegans, but high degrees of in vitro PEC invasion, likely leading to retention of anti-phagocytic activity against D. discoideum predation.

Burkholderia pseudomallei thrives in soil at depths of 30–60 cm. In addition to physical (soil properties and particle sizes) and chemical (pH, metal content, and pollutants) parameters, soil-living amoebae or nematodes inhibit the growth of B. pseudomallei in the environment.47 Burkholderia pseudomallei isolates, under distinct pressures of soil or host environments, exhibit evolutionary genetic diversity to facilitate the development of isolates with increased virulence by adaptive selection.7,36 Although we and other laboratories found that STs were not associated with the virulence of B. pseudomallei or disease presentation in animals or humans,48,49 the virulence-related phenotypic colony morphology, the so-called colony morphovars, flagellar expression, and genomic islands varied among B. pseudomallei isolates.7,50 Clinically, pathogenic patterns differed in different melioidosis-endemic areas. For example, additional cases with prostatic abscesses and encephalomyelitis have been detected among Australian melioidosis patients, whereas melioidosis cases with parotid abscesses and hepatosplenic suppuration are often observed in Thailand.1 In Taiwanese isolates, B. pseudomallei can exhibit Trojan horse–like behavior, potentially invading the central nervous system via leukocyte migration in animal models.14,15,44 Nevertheless, B. pseudomallei isolates isolated from Thailand were reported to directly invade the CNS along nerve fibers by inhalation.51 Based on our studies, the attenuated isolates selected by using C. elegans and D. discoideum may contribute to further exploration of disease progression and/or tissue tropism in animal experiments.

Supplemental tables and figures

Acknowledgments:

This work was supported by grants from the Ministry of Science and Technology (MOST 106-2314-B-075B-009-MY3 [Y. S. C.], MOST105-2320-B-017-MY3 [Y. L. C.], and MOST 104-2311-B-017-001- and MOST 105-2628-B-017-001-MY2 [D. W. H.]), and Kaohsiung Veterans General Hospital (VGHKS105-074, VGHKS106-088 and VGHKS107-105 [Y. S. C.]).

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Author Notes

Address correspondence to Yao-Shen Chen, Department of Internal Medicine, Kaohsiung Veterans General Hospital, No. 386, Ta-Chung 1st Rd., Kaohsiung 81346, Taiwan. E-mail: yschen@vghks.gov.tw

Authors’ addresses: Ya-Lei Chen, Duen-Wei Hsu, Jou-An Chen, and Pei-Jyun Shih, Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung, Taiwan, E-mails: dan1001@ms31.hinet.net, duenwei.hsu@gmail.com, kaiko1101@gmail.com, and spg831105@gmail.com. Pei-Tan Hsueh, Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, E-mail: mulberrymonster@gmail.com. Susan Lee, Section of Infectious Disease, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, E-mail: ssjlee@vghks.gov.tw. Hsi-Hsun Lin, Institute of Public Health, National Yang-Ming University, Taipei, Taiwan, E-mail: ed100233@yahoo.com.tw. Yao-Shen Chen, Department of Internal Medicine, National Yang-Ming University, Taipei, Taiwan, E-mail: yschen@vghks.gov.tw.

These authors contributed equally to this work.

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