Marshall Feterl

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.

Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes

This study used a murine model of type 2 diabetes (BKS.Cg-Dock7(m) +/+Lepr(db)/J mice) to investigate the inflammatory and cellular mechanisms predisposing to Burkholderia pseudomallei infection and co-morbid diabetes. Homozygous db/db (diabetic) mice developed extreme obesity, dyslipidaemia and glucose intolerance leading to hyperglycaemia and overt type 2 diabetes. Compared to their heterozygous db/+ (non-diabetic) littermates, diabetic mice rapidly succumbed to subcutaneous B. pseudomallei infection, paralleled by severe hypoglycaemia and increased expression of the proinflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1β, in the spleen, despite comparable bacterial loads in the spleen of non-diabetic mice. Neutrophil oxidative burst and dendritic cell uptake and killing of B. pseudomallei were similar between diabetic and non-diabetic mice. Compared to peritoneal macrophages from non-diabetic mice, macrophages from diabetic mice were unable to contain and kill B. pseudomallei. Functional differences between macrophages of diabetic and non-diabetic mice toward B. pseudomallei may contribute to rapid dissemination and more severe disease progression in hosts with co-morbid type 2 diabetes.

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.

The effect of different Burkholderia pseudomallei isolates of varying levels of virulence on toll-like-receptor expression

The purpose of this investigation was to ascertain the degree of toll-like-receptor (TLR) activation by Burkholderia pseudomallei isolates with varying levels of virulence 2 h post infection. Standard antibiotic protection assays were performed on RAW 264.7 macrophages and peritoneal exudate cells (PEC) challenged with B. pseudomallei. Real-time PCR (RT-PCR) was performed to determine TLR2, TLR4, TLR5 and TLR9 expression. Internalization and killing of bacteria were determined 2h post infection. ELISAs were performed to determine the levels of TNF-alpha from cultured supernatants. Nitrate levels were determined by Griess assays. Up to 2h post infection, B. pseudomallei failed to significantly increase TLR4, TLR5 and TLR9 expression in both cell types. However, TLR2 expression was increased in RAW 264.7 macrophages, irrespective of isolate virulence. The levels of TNF-alpha and nitrate were significantly attenuated in RAW 264.7 macrophages, and no correlation was found between the level of virulence of the infecting strain and TLR expression, bacterial uptake, or killing. The ability of B. pseudomallei to evade detection by macrophages may in part be due to possible signal dampening of TLRs at very early stages of infection.

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.

The Epidemiology of Malaria in Kutubu, Southern Highlands Province, Papua New Guinea, before and during a Private Sector Initiative for Malaria Control

Papua New Guinea (PNG) has a significant malaria burden, is resource constrained, and has isolated populations with limited access to health services. Home-based management is a key element of the national program that supports strategies of early detection, diagnosis and treatment. We describe the epidemiology of malaria near Lake Kutubu in the Southern Highlands Province through reported data on suspected and confirmed malaria in patients accessing public health facilities or using a novel, incentivised, social marketing approach for malaria treatment at the village level. Monthly case data reported by nine health facilities and 14 village-based providers, known as Marasin Stoa Kipas (MSK), were extracted from outpatient registers and MSK malaria case forms. Descriptive statistics of diagnostic use, monthly incidence, test positivity rate and species distribution were estimated. Summary statistics of service delivery demonstrate patient access and diagnostic coverage in program areas. From May 2005 to September 2013, 15,726 individuals were tested with either rapid diagnostic test and/or microscopy at health facilities, and 42% had a positive result for malaria (n = 6604); of these 67.1% (n = 4431) were positive for P. falciparum (alone or mixed) and 32.9% were positive for non-P. falciparum species (alone or mixed). From October 2007 to September 2013, 9687 individuals were tested with either RDT and/or microscopy at MSK sites and 44.2% (n = 4283) tested positive for malaria; of these, 65.3% (n = 2796) were positive for P. falciparum, while 34.7% (n = 1487) were positive for non-P. falciparum species. Up to April 2010 there was an intermittent and upward trend in the reported incidence of all species of confirmed malaria, reaching 50 per 1000 population per month for both sites combined, followed by a steady decline to four per 1000 population per month in 2013, with P. vivax the most common infection. This study is the most recent longitudinal overview of malaria in the Southern Highlands since 2003. It outlines patient access to a community-based model of care. The analysis shows changes in health facility versus MSK use, a strongly decreasing trend in incidence of confirmed malaria from 2010 to 2013, and a shift from predominantly P. falciparum to P. vivax infection.