Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling. An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries. The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei. Melioidosis is a serious infectious disease caused by the Tier 1 selected agent and Gram-negative environmental saprophyte, Burkholderia pseudomallei. The organism is commonly found in soil and water in melioidosis endemic areas. Infection in humans occurs following bacterial inoculation, inhalation or ingestion. There is a striking lack of accurate information on the global risk of melioidosis, something that could be determined from the global distribution of environmental B. pseudomallei. Soil sampling to detect the presence of B. pseudomallei has been ad hoc, poorly standardized, and the available information poorly collated. Negative studies are almost never reported, and there is no published review on this topic. We responded to this problem during the VI^th World Melioidosis Congress held in Townsville, Australia in December 2010 by forming the ‘Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)’. We have since worked together to undertake a systematic review, map the available information, and reach a consensus on low cost methods for the detection of environmental B. pseudomallei. Our goal is to promote the use of these consensus methods and encourage people worldwide to participate in an effort to produce a comprehensive global map of environmental B. pseudomallei.

Mounting evidence implicates the disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, in global amphibian declines and extinctions. While the virulence of this disease has been clearly demonstrated, there is, as yet, no mechanistic explanation for how B. dendrobatidis kills amphibians. To investigate the pathology of chytridiomycosis, blood samples were collected from uninfected, aclinically infected and clinically diseased amphibians and analyzed for a wide range of biochemical and hematological parameters. Here, we show that green tree frogs Litoria caerulea with severe chytridiomycosis had reduced plasma osmolality, sodium, potassium, magnesium and chloride concentrations. Stable plasma albumin, hematocrit and urea levels indicated that hydration status was unaffected, signifying depletion of electrolytes from circulation rather than dilution due to increased water uptake. We suggest that B. dendrobatidis kills amphibians by disrupting normal epidermal functioning, leading to osmotic imbalance through loss of electrolytes. Determining how B. dendrobatidis kills amphibians is fundamental to understanding the host-pathogen relationship and thus the population declines attributed to B. dendrobatidis. Understanding the mechanisms of mortality may also explain interspecific variation in susceptibility to chytridiomycosis.

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 bb₁₂, bb₁₄ and bb₁₆ 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(Me₄phen)₃]²⁺ showed equal activity (on a mole basis) against S. aureus compared with the Rubb₁₂, Rubb₁₄ and Rubb₁₆, 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(bb₁₆)]²⁺ was significantly less active than Rubb₁₆, highlighting the importance of the dinuclear structure. Preliminary toxicity assays were also carried out for the ΔΔ isomers of Rubb₇, Rubb₁₀, Rubb₁₂ and Rubb₁₆ 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 HC₅₀ and IC₅₀ values 100-fold higher than the MIC for the complex that showed the best potential – ΔΔ-Rubb₁₂.

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(Me₄phen)₃]²⁺ and [Ru(phen)₂(bb₇)]²⁺ against Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli and Pseudomonas aeruginosa. 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. Rubb₁₂ and Rubb₁₆ 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. Rubb₁₆ 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 Rubb₁₂ and Rubb₁₆ 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 Rubb₁₆, and tentatively suggested that the ruthenium complex is localized in the bacteria. The inert dinuclear ruthenium(II) complexes Rubb₁₂ and Rubb₁₆ 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.

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.

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-Rubb₁₂ 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-bb₁₂}]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)(Me₂bpy)Cl]Cl₂ (where Me₂bpy = 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.