Talaromyces (Penicillium) marneffei is an emerging opportunistic pathogen associated with HIV infection, particularly in Southeast Asia and southern China. The rapid uptake and killing of T. marneffei conidia by phagocytic cells along with the effective induction of an inflammatory response by the host is essential for disease control. T. marneffei produces a number of different laccases linked to fungal virulence. To understand the role of the various laccases in T. marneffei, laccase-encoding genes were investigated. Targeted single, double and triple gene deletions of laccases encoding lacA, lacB, and lacC showed no significant phenotypic effects suggesting redundancy of function. When a fourth laccase-encoding gene, pbrB, was deleted in the DlacA DlacB DlacC background, the quadruple mutant displayed delayed conidiation and the conidia were more sensitive to H 2 O 2 , sodium dodecyl sulfate (SDS), and antifungal agents than wild-type and other transformants. Conidia of the quadruple mutant showed marked differences in their interaction with the human monocyte cell line, THP-1 such that phagocytosis was significantly higher when compared with the wild-type at one and 2 hours of incubation while the phagocytic index was significantly different from 15 to 120 minutes. In addition, killing of the quadruple mutant by THP-1 cells was more efficient at 2 and 4 hours of incubation. The levels of the proinflammatory cytokines TNF-a, IL-1b and IL-6 from THP-1 cells infected with the quadruple mutant were also significantly increased in comparison with wild-type. The results demonstrate that production of laccases by T. marneffei actually promotes the pathogen's resistance to innate host defenses.

The biosynthesis of melanin has been linked with virulence in diverse pathogenic fungi. Penicillium marneffei, a dimorphic fungus, is capable of melanization in both mycelial and yeast phases, and the pigment may be produced during infection to protect the fungus from the host immune system. To investigate the impact of yeast morphological transformation on antifungal susceptibility, P. marneffei was cultured on various media including minimal medium, 1% tryptone, brain heart infusion broth, and malt extract broth by using the standardized susceptibility protocol (the M27-A protocol, RPMI medium) for yeasts. We also investigated whether P. marneffei melanization affected its susceptibility to antifungal drugs by adding L-DOPA into culture broths. There were no differences in the minimum inhibitory concentrations (MICs) of P. marneffei yeast cells previously grown in various culture broths with or without L-DOPA using the M27A protocol (into which no melanin substrate can be added due to a rapid color change of the RPMI medium to black) for testing amphotericin B, clotrimazole, fluconazole, itraconazole and ketoconazole. However, both melanized and non-melanized P. marneffei displayed increased resistance to antifungal drugs when L-DOPA was added into a selected assay medium, 0.17% yeast nitrogen base, 2% glucose, and 1.5% agar. Hence, active melanin formation appears to protect P. marneffei by enhancing its resistance to antifungal drugs.

Background:In order to the growing frequency of extendedspectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae, treatment of such infections requires rapid and reliable detection of this enzyme.This study aims to develop the rapid-simple-ESBL detection in these organisms by comparing with the double-disc synergy test/combined disc test.Methods: One hundred and forty-six isolates ofE. coliand 150 isolates ofK. pneumoniaeisolated from the clinical specimens were included in this study. The rapid-simple-ESBL (RS-ESBL) test was performed in phenol red glucose broth containing ceftazidime(CAZ) or cefotaxime(CTX) (2 g/ml) with or without clavulanic acid in microtiter plate. And the incubation period of this test was 4 hours. All of them were also tested for ESBL producer by double-disc synergy test/combined disc test.Results: Among 96 ESBL-producing E. coli isolates, 94(97.9%) and 95(99.0%) were identified as ESBL-positive by CAZ RS-ESBL and CTX RS-ESBL, respectively when compared with double-disc synergy test/combined disc test. Of 100 ESBL-producingK. pneumoniae, 97(97.0%) were ESBL-positive by both CAZ RS-ESBL and CTX RS-ESBL tests. All of the 100 non-ESBL producers were negative by CTX RS-ESBL test. Conclusion:The RS-ESBL test was proved to be a reliable, simple and rapid method for detecting ESBL in E. coli and K. pneumoniae.