Expanding the Burkholderia pseudomallei Complex with the Addition of Two Novel Species: Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov.

Distinct Burkholderia strains were isolated from soil samples collected in tropical northern Australia (Northern Territory and the Torres Strait Islands, Queensland). Phylogenetic analysis of 16S rRNA and whole genome sequences revealed these strains were distinct from previously described Burkholderia species and assigned them to two novel clades within the B. pseudomallei complex (Bpc). Because average nucleotide identity and digital DNA-DNA hybridization calculations are consistent with these clades representing distinct species, we propose the names Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov. Strains assigned to B. mayonis sp. nov. include type strain BDU6T (=TSD-80; LMG 29941; ASM152374v2) and BDU8. Strains assigned to B. savannae sp. nov. include type strain MSMB266T (=TSD-82; LMG 29940; ASM152444v2), MSMB852, BDU18, and BDU19. Comparative genomics revealed unique coding regions for both putative species, including clusters of orthologous genes associated with phage. Type strains of both B. mayonis sp. nov. and B. savannae sp. nov. yielded biochemical profiles distinct from each other and from other species in the Bpc, and profiles also varied among strains within B. mayonis sp. nov. and B. savannae sp. nov. Matrix-assisted laser desorption ionization time-of-flight (MLST) analysis revealed a B. savannae sp. nov. cluster separate from other species, whereas B. mayonis sp. nov. strains did not form a distinct cluster. Neither B. mayonis sp. nov. nor B. savannae sp. nov. caused mortality in mice when delivered via the subcutaneous route. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species currently within the Bpc. IMPORTANCEBurkholderia species can be important sources of novel natural products, and new species are of interest to diverse scientific disciplines. Although many Burkholderia species are saprophytic, Burkholderia pseudomallei is the causative agent of the disease melioidosis. Understanding the genomics and virulence of the closest relatives to B. pseudomallei, i.e., the other species within the B. pseudomallei complex (Bpc), is important for identifying robust diagnostic targets specific to B. pseudomallei and for understanding the evolution of virulence in B. pseudomallei. Two proposed novel species, B. mayonis sp. nov. and B. savannae sp. nov., were isolated from soil samples collected from multiple locations in northern Australia. The two proposed species belong to the Bpc but are phylogenetically distinct from all other members of this complex. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species within this significant complex of bacteria that are available for future studies.

Melioidosis in Papua New Guinea and Oceania.

Melioidosis has only been sporadically reported throughout Melanesia and the Pacific region since the first report from Guam in 1946; therefore, its contribution to the disease burden in this region is largely unknown. However, the outcome of a small number of active surveillance programs, serological surveys, and presumptive imported cases identified elsewhere provide an insight into its epidemiology and potential significance throughout the region. Both clinical cases and environmental reservoirs have been described from the rural district of Balimo in the Western Province of Papua New Guinea and from the Northern Province of New Caledonia. In both these locations the incidence of disease is similar to that described in tropical Australia and Burkholderia pseudomallei isolates are also phylogenetically linked to Australian isolates. Serological evidence and presumptive imported cases identified elsewhere suggest that melioidosis exists in other countries throughout the Pacific. However, the lack of laboratory facilities and clinical awareness, and the burden of other infections of public health importance such as tuberculosis, contribute to the under-recognition of melioidosis in this region.

Probing the pharmacokinetics of cucurbit[7, 8 and 10]uril: and a dinuclear ruthenium antimicrobial complex encapsulated in cucurbit[10]uril.

The relatively non-toxic family of cucurbit[n]uril, Q[n], have shown considerable potential in vitro as drug delivery agents, with only a few examples of pharmacokinetic (PK) studies for drug⊂Q[n]. Drug-free Q[n] PK studies are the next step in determining the pharmacological applicability in their drug delivery potential. The results for the first PK and bio-distribution of drug-free 14C-Q[7] are described for administration via intravenous (i.v.) and intraperitoneal (i.p.) dosing. A study of oral administration of drug-free 14C-Q[8] has also been undertaken to determine the time course for the gastrointestinal tract (GIT), absorption and subsequent bio-distribution. Q[10], a potential drug carrier for larger drugs, was evaluated for its effect on the PK profile of a dinuclear ruthenium complex (Rubb12), a potential antimicrobial agent. The Rubb12⊂Q[10] complex and free Rubb12 were administered by i.v. to determine differences in Rubb12 plasma concentrations and organ accumulation. Interestingly, the PK profiles and bio-distribution observed for Q[7] showed similarities to those of Rubb12⊂Q[10]. Drug-free Q[7] has a relatively fast plasma clearance and a generally low organ accumulation except for the kidneys. Drug-free Q[8] showed a low absorption from the GIT into the blood stream but the small percentage absorbed reflected the organ accumulation of Q[7]. These results provide a better understanding of the probable PK profile and bio-distribution for a drug⊂Q[n] through the influence of the drug delivery vehicle and the positive clearance of drug-free Q[n] via the kidneys supports its potential value in future drug delivery applications.

Oligonuclear polypyridylruthenium(II) complexes: selectivity between bacteria and eukaryotic cells.

OBJECTIVES: The objectives of this study were to: (i) determine the in vitro activities of a series of di-, tri- and tetra-nuclear ruthenium complexes (Rubbn, Rubbn-tri and Rubbn-tetra) against a range of Gram-positive and -negative bacteria and compare the antimicrobial activities with the corresponding toxicities against eukaryotic cells; and (ii) compare MIC values with achievable in vivo serum concentrations for the least toxic ruthenium complex. METHODS: The in vitro activities were determined by MIC assays and time-kill curve experiments, while the toxicities of the ruthenium complexes were determined using the Alamar blue cytotoxicity assay. A preliminary pharmacokinetic study was undertaken to determine the Rubb12 serum concentration in mice as a function of time after administration. RESULTS: Rubb12, Rubb12-tri and Rubb12-tetra are highly active, with MIC values of 1-2 mg/L (0.5-1.5 µM) for a range of Gram-positive strains, but showed variable activities against a panel of Gram-negative bacteria. Time-kill experiments indicated that Rubb12, Rubb12-tri and Rubb12-tetra are bactericidal and kill bacteria within 3-8 h. The di-, tri- and tetra-nuclear complexes were ∼50 times more toxic to Gram-positive bacteria and 25 times more toxic to Gram-negative strains, classified as susceptible, than to liver and kidney cells. Preliminary pharmacokinetic experiments established that serum concentrations higher than MIC values can be obtained for Rubb12 with an administered dose of 32 mg/kg. CONCLUSIONS: The ruthenium complexes, particularly Rubb12, have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complex can be readily further modified in order to increase the selectivity for bacteria over eukaryotic cells.

Dinuclear ruthenium(ii) complexes containing one inert metal centre and one coordinatively-labile metal centre: syntheses and biological activities.

A series of non-symmetric dinuclear polypyridylruthenium(ii) complexes (Rubbn-Cl) that contain one inert metal centre and one coordinatively-labile metal centre, linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ligand ("bbn" for n = 7, 12 and 16), have been synthesised and their potential as antimicrobial agents examined. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria--Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The Rubbn-Cl complexes displayed good antimicrobial activity, with Rubb12-Cl being the most active complex against both Gram-positive and Gram-negative strains. Interestingly, Rubb7-Cl was found to be eight- and sixteen-fold more active towards E. coli than against S. aureus and MRSA, respectively. The cytotoxicities of the Rubbn-Cl complexes against three eukaryotic cell lines--two kidney cell lines (BHK and HEK-293) and one liver cell line (HepG2)--were examined. The Rubbn-Cl complexes were found to be considerably less toxic towards eukaryotic cells than S. aureus, MRSA and E. coli, with Rubb12-Cl being thirty- to eighty-times more toxic to the bacteria than to BHK, HEK-293 or HepG2 cells. Unexpectedly, Rubb7-Cl was far more toxic to HepG2 cells (24 h-IC50 = 3.7 µM) and far less toxic to BHK cells (24 h-IC50 = 238 µM) than the Rubb12-Cl and Rubb16-Cl complexes. In order to understand the unexpected large differences in the cytotoxicities of the Rubbn-Cl complexes towards eukaryotic cells, a confocal microscopic study of their intracellular localisation was undertaken. The results suggest that the observed cytotoxicity might be related to the extent of DNA binding.

Burkholderia pseudomallei is frequently detected in groundwater that discharges to major watercourses in northern Australia.

Burkholderia pseudomallei is the environmental bacterium that causes the serious disease melioidosis. Recently, a high prevalence of viable B. pseudomallei was reported from natural groundwater seeps around Castle Hill, a clinical focus of melioidosis in Townsville, Australia. This study sought to expand previous findings to determine the extent of B. pseudomallei in more diverse natural groundwater seeps in northern Queensland to ascertain if the presence of the organism in groundwater on Castle Hill was an isolated occurrence. Analysis of water samples (n = 26) obtained from natural groundwater seeps following an intensive rainfall event in the Townsville region determined the presence of B. pseudomallei DNA in duplicates of 18 samples (69.2 % [95 % CI, 51.5 to 87.0]). From 26 water samples, a single isolate of B. pseudomallei was recovered despite plating of both pre-enriched samples and original water samples onto selective media, indicating that the sensitivity of these molecular techniques far exceeds culture-based methods. Furthermore, the identification of new environments endemic for melioidosis may be more effectively determined by analysing surface groundwater seeps than by the analysis of random soil samples. This study suggests that a higher incidence of melioidosis following monsoonal rains may be partially the result of exposure to groundwater sources carrying B. pseudomallei, and that modifications to public health messages in endemic regions may be warranted. Moreover, these findings have implications for predictive models of melioidosis, effective models requiring consideration of topographical and surface hydrological data.

Environmental Attributes Influencing the Distribution of Burkholderia pseudomallei in Northern Australia.

Factors responsible for the spatial and temporal clustering of Burkholderia pseudomallei in the environment remain to be elucidated. Whilst laboratory based experiments have been performed to analyse survival of the organism in various soil types, such approaches are strongly influenced by alterations to the soil micro ecology during soil sanitisation and translocation. During the monsoonal season in Townsville, Australia, B. pseudomallei is discharged from Castle Hill (an area with a very high soil prevalence of the organism) by groundwater seeps and is washed through a nearby area where intensive sampling in the dry season has been unable to detect the organism. We undertook environmental sampling and soil and plant characterisation in both areas to ascertain physiochemical and macro-floral differences between the two sites that may affect the prevalence of B. pseudomallei. In contrast to previous studies, the presence of B. pseudomallei was correlated with a low gravimetric water content and low nutrient availability (nitrogen and sulphur) and higher exchangeable potassium in soils favouring recovery. Relatively low levels of copper, iron and zinc favoured survival. The prevalence of the organism was found to be highest under the grasses Aristida sp. and Heteropogon contortus and to a lesser extent under Melinis repens. The findings of this study indicate that a greater variety of factors influence the endemicity of melioidosis than has previously been reported, and suggest that biogeographical boundaries to the organisms' distribution involve complex interactions.

Mononuclear Polypyridylruthenium(II) Complexes with High Membrane Permeability in Gram-Negative Bacteria-in particular Pseudomonas aeruginosa.

Ruthenium(II) complexes containing the tetradentate ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ("bbn "; n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bbn )](2+) (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-α-[Ru(phen)(bb12 )](2+) species showed good activity against Gram-negative species. In particular, the cis-α-[Ru(phen)(bb12 )](2+) complex was two to four times more active than the cis-ß-[Ru(phen)(bb12 )](2+) complex against the Gram-negative strains. The cis-α- and cis-ß-[Ru(phen)(bb12 )](2+) complexes readily accumulated in the bacteria but, significantly, showed the highest level of uptake in Pseudomonas aeruginosa. Furthermore, the accumulation of the cis-α- and cis-ß-[Ru(phen)(bb12 )](2+) complexes in P. aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-α-[Ru(phen)(bb12 )](2+) complex into live P. aeruginosa was confirmed by using fluorescence microscopy. The water/octanol partition coefficients (log P) were determined to gain understanding of the relative cellular uptake. The cis-α- and cis-ß-[Ru(phen)(bbn )](2+) complexes exhibited relatively strong binding to DNA (Kb ≈10(6) M(-1) ), but no significant difference between the geometric isomers was observed.

Seroepidemiology of melioidosis in children from a remote region of Papua New Guinea.

BACKGROUND: The Balimo region in Papua New Guinea has previously been identified as melioidosis-endemic with a predilection for children. Where health resources are scarce, seroepidemiology can be used to assess exposure to Burkholderia pseudomallei and therefore risk of acquiring melioidosis. METHODS: Logistic regression was used to determine associations between indirect haemagglutination assay (IHA) seroreactivity with environmental and demographic/cultural factors to aid in determining risk factors associated with exposure to B. pseudomallei in children. RESULTS: Of the 968 participants, 92.9% (899/968) were children, representing the majority of the community school population in the immediate Balimo region. Of these, 24.6% (221/899) were seropositive. Bathing in the lagoon (OR=2.679), drinking from the well or lagoon (OR=1.474), and being a member of the Siboko (OR=1.914) or Wagumisi (OR=1.942) clans were significantly associated with seropositivity. In the multivariate analysis, drinking from a well or lagoon (OR=1.713), and the Siboko (OR=2.341) and Wabadala (OR=2.022) clans were associated with seropositivity. CONCLUSIONS: This study in children supports observations that interactions with groundwater in this region are risk factors in acquiring melioidosis. Public health measures intended to limit this exposure may help reduce the risk of acquiring melioidosis in this remote community. Associations with clan structure may provide more cultural specific insights, however this requires further elucidation.

Tri- and tetra-nuclear polypyridyl ruthenium(II) complexes as antimicrobial agents.

A series of inert tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ligand ("bb(n)" for n = 10, 12 and 16) have been synthesised and their potential as antimicrobial agents examined. Due to the modular nature of the synthesis of the oligonuclear complexes, it was possible to make both linear and non-linear tetranuclear ruthenium species. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria--Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In order to gain an understanding of the relative antimicrobial activities, the cellular uptake and water-octanol partition coefficients (log P) were determined for a selection of the ruthenium complexes. Although the trinuclear complexes were the most lipophilic based upon log P values and showed the greatest cellular uptake, the linear tetranuclear complexes were generally more active, with MIC values <1 µM against the Gram positive bacteria. Similarly, although the non-linear tetranuclear complexes were slightly more lipophilic and were taken up to a greater extent by the bacteria, they were consistently less active than their linear counterparts. Of particular note, the cellular accumulation of the oligonuclear ruthenium complexes was greater in the Gram negative strains compared to that in the Gram positive S. aureus and MRSA. The results demonstrate that the lower antimicrobial activity of polypyridylruthenium(II) complexes towards Gram negative bacteria, particularly P. aeruginosa, is not strongly correlated to the cellular accumulation but rather to a lower intrinsic ability to kill the Gram negative cells.

Dinuclear polypyridylruthenium(II) complexes: flow cytometry studies of their accumulation in bacteria and the effect on the bacterial membrane.

OBJECTIVES: To determine the energy dependency of and the contribution of the membrane potential to the cellular accumulation of the dinuclear complexes [{Ru(phen)2}2{µ-bbn}](4+) (Rubbn) and the mononuclear complexes [Ru(Me4phen)3](2+) and [Ru(phen)2(bb7)](2+) in Staphylococcus aureus and Escherichia coli, and to examine their effect on the bacterial membrane. METHODS: The accumulation of the ruthenium complexes in bacteria was determined using flow cytometry at a range of temperatures. The cellular accumulation of the ruthenium complexes was also determined in cells that had been incubated with the metal complexes in the presence or absence of metabolic stimulators or inhibitors and/or commercial dyes to determine the membrane potential or membrane permeability. RESULTS: The accumulation of ruthenium complexes in the two bacterial strains was shown to increase with increasing incubation temperature, with the relative increase in accumulation greater with E. coli, particularly for Rubb12 and Rubb16. No decrease in accumulation was observed for Rubb12 in ATP-inhibited cells. While carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did depolarize the cell membrane, no reduction in the accumulation of Rubb12 was observed; however, all ruthenium complexes, when incubated with S. aureus at concentrations twice their MIC, depolarized the membrane to a similar extent to CCCP. Except for the mononuclear complex [Ru(Me4phen)3](2+), incubation of any of the other ruthenium complexes allowed a greater quantity of the membrane-impermeable dye TO-PRO-3 to be taken up by S. aureus. CONCLUSIONS: The results indicate that the potential new antimicrobial Rubbn complexes enter the cell in an energy-independent manner, depolarize the cell membrane and significantly permeabilize the cellular membrane.

Protein binding by dinuclear polypyridyl ruthenium(II) complexes and the effect of cucurbit[10]uril encapsulation.

The effect of human serum on the minimum inhibitory/bactericidal concentrations of the potential antimicrobial agents ΔΔ-[{Ru(phen)2}2(µ-bb(n))](4+) {ΔΔ-Rubb(n); where phen = 1,10-phenanthroline, bb(n) = 1,n-bis[4(4'-methyl-2,2'-bipyridyl)]-alkane for n = 12 and 16} against four strains of bacteria--Gram positive Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli and Pseudomonas aeruginosa--has been determined. The results demonstrated that the ruthenium(ii) complexes have significantly decreased in vitro activity in serum. Fluorescence spectroscopy was used to confirm that the decrease in antimicrobial activity was due to the strong binding of the ruthenium complexes with the serum proteins human serum albumin (HSA) and transferrin. A series of ruthenium complexes showed stronger binding to HSA than apo-transferrin but comparable or less than with holo-transferrin, with the binding affinity to all three proteins decreasing in the order trinuclear > dinuclear > mononuclear. The dinuclear complex ΔΔ-Rubb12 displaced warfarin from HSA, tentatively suggesting that the ruthenium complexes bind at or near the warfarin-binding site, Sudlow's site 1. The binding of ΔΔ-Rubb12 and ΔΔ-Rubb16 to the macrocyclic host molecule cucurbit[10]uril (Q[10]) was examined by NMR spectroscopy. The large upfield (1)H NMR chemical shift changes observed for the methylene protons in the bridging ligands upon addition of Q[10], coupled with the observation of a range of intermolecular ROEs in ROESY spectra, indicated that the dinuclear complexes bound Q[10] with the bridging ligand within the cavity and the metal centres positioned outside the portals. NMR and fluorescence spectroscopy demonstrated that the Q[10]-encapsulated ruthenium complexes directly bound HSA, and with similar affinity to the corresponding free metal complexes.

Chlorido-containing ruthenium(II) and iridium(III) complexes as antimicrobial agents.

A series of polypyridyl-ruthenium(II) and -iridium(III) complexes that contain labile chlorido ligands, [{M(tpy)Cl}(2){µ-bb(n)}](2/4+) {Cl-Mbb(n); where M = Ru or Ir; tpy = 2,2':6',2''-terpyridine; and bb(n) = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 or 16)} have been synthesised and their potential as antimicrobial agents examined. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the series of metal complexes against four strains of bacteria - Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) - have been determined. All the ruthenium complexes were highly active and bactericidal. In particular, the Cl-Rubb(12) complex showed excellent activity against all bacterial cell lines with MIC values of 1 µg mL(-1) against the Gram positive bacteria and 2 and 8 µg mL(-1) against E. coli and P. aeruginosa, respectively. The corresponding iridium(III) complexes also showed significant antimicrobial activity in terms of MIC values; however and surprisingly, the iridium complexes were bacteriostatic rather than bactericidal. The inert iridium(III) complex, [{Ir(phen)(2)}(2){µ-bb(12)}](6+) {where phen = 1,10-phenanthroline) exhibited no antimicrobial activity, suggesting that it could not cross the bacterial membrane. The mononuclear model complex, [Ir(tpy)(Me(2)bpy)Cl]Cl(2) (where Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine), was found to aquate very rapidly, with the pK(a) of the iridium-bound water in the corresponding aqua complex determined to be 6.0. This suggests the dinuclear complexes [Ir(tpy)Cl}(2){µ-bb(n)}](4+) aquate and deprotonate rapidly and enter the bacterial cells as 4+ charged hydroxo species.

Sago haemolytic disease: towards understanding a novel food-borne toxicosis.

Sago haemolytic disease is a rare but sometimes fatal disease found primarily in the coastal regions of Papua New Guinea and among groups in which sago is a primary source of carbohydrate. It has been known since 1961 and fungi consistently have been suspected of being involved. Investigations carried out on stored sago and samples recovered from poisoning episodes have failed to indicate the consistent presence of mycotoxins. However, fungi (especially Aspergillus, Fusarium, Penicillium, Trichoderma) with strong haemolytic activity have been associated with sago, particularly when stored in open-weave baskets and sago-leaf-wrapped bundles. The haemolytic activity has been attributed to fatty acids (principally oleic, palmitic, linoleic) contained primarily in the fungal hyphae. It is hypothesized that when these acids are released through hyphal breakdown during digestion and are present in individuals with a low serum albumin level, free fatty acid excess occurs resulting in red cell membrane destruction and intravascular haemolysis. In extreme cases, blood transfusion is required. Methods of storage providing high levels of access to oxygen favour the development of fungi: eg, leaf-encased bundles and open-weave storage favour growth over that seen in starch stored under water, such as in earthen vessels. Ensuring storage does not exceed 3-4 weeks, encouraging anaerobic conditions of the starch and maintaining protein nutrition in communities where sago is relied upon should alleviate outbreak episodes.

In vitro susceptibility and cellular uptake for a new class of antimicrobial agents: dinuclear ruthenium(II) complexes.

OBJECTIVES: To determine the in vitro susceptibility and cellular uptake for a series of dinuclear ruthenium(II) complexes [{Ru(phen)(2)}(2){µ-bb(n)}](4+) (Rubb(n)), and the mononuclear complexes [Ru(Me(4)phen)(3)](2+) and [Ru(phen)(2)(bb(7))](2+) against Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli and Pseudomonas aeruginosa. METHODS: The in vitro susceptibility was determined by MIC and MBC assays, and time-kill curve experiments, while the cellular uptake was evaluated by monitoring the fluorescence of the complexes remaining in the supernatant of the cultures after incubation for various periods of time, flow cytometry and confocal microscopy. RESULTS: Rubb(12) and Rubb(16) are highly active, with MIC and MBC values of 1-2 mg/L (0.5-1 µM) for the two Gram-positive strains and 2-4 mg/L for E. coli and 16-32 mg/L for P. aeruginosa. Rubb(16) showed equal or better activity (on a molar basis) to gentamicin and ampicillin for all strains apart from P. aeruginosa. The relative MBC to MIC values indicated that Rubb(12) and Rubb(16) are bactericidal, and from the time-kill curve experiments, the ruthenium complexes can kill the bacteria within 2-6 h. The cellular uptake studies demonstrated that the observed antimicrobial activity is correlated with the level of uptake of the ruthenium complexes. Confocal microscopy confirmed the cellular uptake of Rubb(16), and tentatively suggested that the ruthenium complex is localized in the bacteria. CONCLUSIONS: The inert dinuclear ruthenium(II) complexes Rubb(12) and Rubb(16) have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complexes can be readily further modified in order to increase their selectivity for bacteria over human cells.

Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications.

The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

The antimicrobial activity of inert oligonuclear polypyridylruthenium(II) complexes against pathogenic bacteria, including MRSA.

The minimum inhibitory concentrations (MIC) of a series of synthetic inert polypyridylruthenium(II) complexes against four strains of bacteria--Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa)--have been determined. The results demonstrate that for the dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)(2)}(2){µ-bb(n)}](4+) {where phen = 1,10-phenanthroline; bb(n) = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16)} the complexes linked by the bb(12), bb(14) and bb(16) ligands are highly active, with MIC values of 1 µg mL(-1) against both S. aureus and MRSA, and 2-4 and 8-16 µg mL(-1) against E. coli and P. aeruginosa, respectively. The mononuclear complex [Ru(Me(4)phen)(3)](2+) showed equal activity (on a mole basis) against S. aureus compared with the Rubb(12), Rubb(14) and Rubb(16), but was considerably less active against MRSA and the two Gram negative bacteria. For the dinuclear Rubb(n) family of complexes, the antimicrobial activity was related to the octanol-water partition coefficient (logP). However, the highly lipophilic mononuclear complex Δ-[Ru(phen)(2)(bb(16))](2+) was significantly less active than Rubb(16), highlighting the importance of the dinuclear structure. Preliminary toxicity assays were also carried out for the ΔΔ isomers of Rubb(7), Rubb(10), Rubb(12) and Rubb(16) against two human cells lines, fresh red blood cells and THP-1 cells. The results showed that the dinuclear ruthenium complexes are significantly less toxic to human cells compared to bacterial cells, with the HC(50) and IC(50) values 100-fold higher than the MIC for the complex that showed the best potential--ΔΔ-Rubb(12).

Antibiosis of Burkholderia ubonensis againist Burkholderia pseudomallei, the causative agent for melioidosis.

Melioidosis, caused by Burkholderia pseudomallei, is an enigmatic infectious disease that afflicts individuals in many tropical and developing regions. Treatment is hampered by the organism's innate antibiotic resistance and the disease's non-pathognomic presentation. Recently, added attention has been given to this organism due to its classification as a potential biowarfare agent. Therefore, methods of preventing acquisition of infection are needed. We investigated antagonism between Burkholderia spp and B. pseudomallei derived from the same ecological niche in a melioidosis endemic region in Papua New Guinea. Isolates of environmentally derived non-pseudomallei Burkholderia spp (n=16) were screened for antibiosis against 27 B. pseudomallei isolates. Three isolates subsequently identified as B. ubonensis produced specific antagonistic activity against all B. pseudomallei isolates tested. The antagonistic compound in a cell-free state was obtained from a representative producing strain, with subsequent biological characterization revealing a pepsin sensitive peptide moiety consistent with a bacteriocin-like compound. To our knowledge, this is the first report of antagonistic activity demonstrated by near-neighbor Burkholderia against B. pseudomallei. This antagonism may be important in the micro-ecology of B. pseudomallei, and could also have application in the biocontrol of this pathogen.

Is Penicillium citrinum implicated in sago hemolytic disease?

Sago hemolytic disease (SHD) is an acute hemolytic syndrome affecting rural Papua New Guineans who depend on the starch of Metroxylon sagu as a staple carbohydrate. It is a suspected mycotoxicosis associated with fungal succession in stored and perhaps poorly fermented sago. Despite a mortality rate of approximately 25%, little is know about the disease. Recent studies have identified Penicillium citrinum as a possible candidate in the etiology of SHD. This is based on the frequency of isolation from sago starch and the hemolytic nature of the organism as demonstrated when cultured on sheep and human blood agar. A highly non-polar lipophilic P. citrinum fraction from C18 solid phase extraction demonstrated high hemolytic activity in a semi-quantitative assay using both mouse and human erythrocytes. When the red cell membrane proteins were subjected to sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, cleavage of protein band 3 and spectrin was demonstrated. This breach of major structural red cell proteins is consistent with the severe hemolysis found in vivo. Our findings warrant further investigation into the hemolytic activity of P. citrinum and its role as the etiological agent of SHD.

Haemolytic fungi isolated from sago starch in Papua New Guinea.

Sago haemolytic disease (SHD) is a rare but often fatal illness linked to consumption of stale sago starch in Papua New Guinea. Although the aetiology of SHD remains unknown, mycotoxins are suspected. This study investigated whether fungi isolated from Papua New Guinean sago starch were haemolytic. Filamentous fungi and yeasts from sago starch were grown on sheep blood agar and some on human blood agar. Clear haemolytic activity was demonstrated by 55% of filamentous fungal isolates, but not by yeasts. A semi-quantitative bioassay was developed involving incubation of human erythrocytes with fungal extracts. Extracts of cultures of Penicillium, Aspergillus and Fusarium all caused rapid haemolysis in the bioassay. Partial fractionation of extracts suggested that both polar and non-polar haemolytic components had haemolytic activity in vitro. Further work is warranted to identify these metabolites and determine if they play a role in SHD.