Profiling of Bacterial and Fungal Microbial Communities in Cystic Fibrosis Sputum Using RNA.

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect 42 bacterial and fungal genera or species common in cystic fibrosis (CF) sputum and demonstrated the taxon specificity of these probes, as well as a linear response over more than 3 logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString, and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. Compared at the genus level, the relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and with further development, may provide quick and easily interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.IMPORTANCE Here we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

Effect of an oral iron chelator or iron-deficient diets on uroporphyria in a murine model of porphyria cutanea tarda.

UNLABELLED: Porphyria cutanea tarda is a liver disease characterized by elevated hepatic iron and excessive production of uroporphyrin (URO). Phlebotomy is an effective treatment that probably acts by reducing hepatic iron. Here we used Hfe(-/-) mice to compare the effects on hepatic URO accumulation of two different methods of hepatic iron depletion: iron chelation using deferiprone (L1) versus iron-deficient diets. Hfe(-/-) mice in a 129S6/SvEvTac background were fed 5-aminolevulinic acid (ALA), which results in hepatic URO accumulation, and increasing doses of L1 in the drinking water. Hepatic URO accumulation was completely prevented at low L1 doses, which partially depleted hepatic nonheme iron. By histological assessment, the decrease in hepatic URO accumulation was associated with greater depletion of iron from hepatocytes than from Kupffer cells. The L1 treatment had no effect on levels of hepatic cytochrome P4501A2 (CYP1A2). L1 also effectively decreased hepatic URO accumulation in C57BL/6 Hfe(-/-) mice treated with ALA and a CYP1A2 inducer. ALA-treated mice maintained on defined iron-deficient diets, rather than chow diets, did not develop uroporphyria, even when the animals were iron-supplemented either directly in the diet or by iron dextran injection. CONCLUSION: The results suggest that dietary factors other than iron are involved in the development of uroporphyria and that a modest depletion of hepatocyte iron by L1 is sufficient to prevent URO accumulation.

Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda.

UNLABELLED: Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3',4,4',5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. CONCLUSION: In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.