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Diagnostic errors can occur, in infectious diseases, when anti-microbial immune responses involve several temporal scales. When responses span from nanosecond to week and larger temporal scales, any pre-selected temporal scale is likely to miss some (faster or slower) responses. Hoping to prevent diagnostic errors, a pilot study was conducted to evaluate a four-dimensional (4D) method that captures the complexity and dynamics of infectious diseases.Leukocyte-microbial-temporal data were explored in canine and human (bacterial and/or viral) infections, with: (i) a non-structured approach, which measures leukocytes or microbes in isolation; and (ii) a structured method that assesses numerous combinations of interacting variables. Four alternatives of the structured method were tested: (i) a noise-reduction oriented version, which generates a single (one data point-wide) line of observations; (ii) a version that measures complex, three-dimensional (3D) data interactions; (iii) a non-numerical version that displays temporal data directionality (arrows that connect pairs of consecutive observations); and (iv) a full 4D (single line-, complexity-, directionality-based) version.In all studies, the non-structured approach revealed non-interpretable (ambiguous) data: observations numerically similar expressed different biological conditions, such as recovery and lack of recovery from infections. Ambiguity was also found when the data were structured as single lines. In contrast, two or more data subsets were distinguished and ambiguity was avoided when the data were structured as complex, 3D, single lines and, in addition, temporal data directionality was determined. The 4D method detected, even within one day, changes in immune profiles that occurred after antibiotics were prescribed.Infectious disease data may be ambiguous. Four-dimensional methods may prevent ambiguity, providing earlier, in vivo, dynamic, complex, and personalized information that facilitates both diagnostics and selection or evaluation of anti-microbial therapies.

Opportunistic fungal pathogens may cause an array of superficial infections or serious invasive infections, especially in immunocompromised patients. Cryptococcus neoformans is a pathogen causing cryptococcosis in HIV/AIDS patients, but treatment is limited due to the relative lack of potent antifungal agents. Photodynamic inactivation (PDI) uses the combination of non-toxic dyes called photosensitizers and harmless visible light, which produces singlet oxygen and other reactive oxygen species that produce cell inactivation and death. We report the use of five structurally unrelated photosensitizers (methylene blue, Rose Bengal, selenium derivative of a Nile blue dye, a cationic fullerene and a conjugate between poly-L-lysine and chlorin(e6)) combined with appropriate wavelengths of light to inactivate C. neoformans. Mutants lacking capsule and laccase, and culture conditions that favoured melanin production were used to probe the mechanisms of PDI and the effect of virulence factors. The presence of cell wall, laccase and melanin tended to protect against PDI, but the choice of the appropriate photosensitizers and dosimetry was able to overcome this resistance.

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

BACKGROUND:Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. METHODOLOGY/PRINCIPAL FINDINGS:In this study we report the identification and characterization of 4',5'-O-dicaffeoylquinic acid (4',5'-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3',5'-ODCQA, 4',5'-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4',5'-ODCQA with pump inhibitory activity whereas 3',5'-ODCQA was ineffective. These initial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. CONCLUSIONS/SIGNIFICANCE:These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4',5'-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.

Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals.