Fecal Excretion of Mycobacterium leprae, Burkina Faso.

Mycobacterium leprae was detected by optical microscopy, fluorescent in situ hybridization, and molecular detection in feces collected for the diagnosis of Entamoeba coli enteritis in a leprosy patient in Burkina Faso. This observation raises questions about the role of fecal excretion of M. leprae in the natural history and diagnosis of leprosy.

Fluorescent Hybridization of Mycobacterium leprae in Skin Samples Collected in Burkina Faso.

Leprosy is caused by Mycobacterium leprae, and it remains underdiagnosed in Burkina Faso. We investigated the use of fluorescent in situ hybridization (FISH) for detecting M. leprae in 27 skin samples (skin biopsy samples, slit skin samples, and skin lesion swabs) collected from 21 patients from Burkina Faso and three from Côte d'Ivoire who were suspected of having cutaneous leprosy. In all seven Ziehl-Neelsen-positive skin samples (four skin biopsy samples and three skin swabs collected from the same patient), FISH specifically identified M. leprae, including one FISH-positive skin biopsy sample that remained negative after testing with PCR targeting the rpoB gene and with the GenoType LepraeDR assay. Twenty other skin samples and three negative controls all remained negative for Ziehl-Neelsen staining, FISH, and rpoB PCR. These data indicate the usefulness of a microscopic examination of skin samples after FISH for first-line diagnosis of cutaneous leprosy. Accordingly, FISH represents a potentially useful point-of-care test for the diagnosis of cutaneous leprosy.

Monitoring of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris (synonym Candida zemplinina) populations during alcoholic fermentation by fluorescence in situ hybridization.

Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15 °C and 25 °C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated.

Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization.

Real-time detection of microorganisms involved in complex microbial process, such as wine fermentations, and evaluation of their physiological state is crucial to predict whether or not those microbial species will be able to impact the final product. In the present work we used a direct live/dead staining (LDS) procedure combined with fluorescence in situ hybridization (FISH) to simultaneously assess the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) during fermentations performed with single and mixed cultures. The population evolution of both yeasts was determined by plating and by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since the FISH method involves the permeabilization of the cell membrane prior to hybridization and that it may influence the free diffusion of PI in and out of the cells, we optimized the concentration of this dye (0.5 µg of PI per 10(6) cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by hemocytometry and flow cytometry. Results showed that the survival rate of Sc during mixed cultures was high throughout the entire process (60% of viable cells at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead cells at the 3rd day (45 g/l of ethanol) and became completely unculturable at the 4th day. However, under single culture fermentation the survival rate and culturability of Hg decreased at a much slower pace, exhibiting at the 7th day (67 g/l of ethanol) 8.7×10(4) CFU/ml and 85% of dead cells. Thus, our work demonstrated that the LDS-FISH method is able to simultaneously assess the viability and identity of these wine-related yeast species during alcoholic fermentation in a fast and reliable way. In order to validate PI-staining as a viability marker during alcoholic fermentation, we evaluated the effect of ethanol on the membrane permeability of Sc and Hg cells, as well as their capacity to recover membrane integrity after being exposed to different levels of ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to recover membrane integrity after ethanol exposure, Hg cells were not. However, under alcoholic fermentation Sc cells didn't recover membrane integrity after the mid-term (4-5 days) of alcoholic fermentation.

Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR.

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (107-108 cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 108 cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.

Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections.

With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations.

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.

The XIST locus replicates late on the active X, and earlier on the inactive X based on FISH DNA replication analysis of somatic cell hybrids.

We have recently reported results of DNA replication analysis of three X-linked loci (FRAXA, F8C and XIST) on the X chromosomes in male and female fibroblasts using fluorescence in situ hybridization (FISH) (1). Although our findings that XIST replicates later on the active X than on the inactive X are similar to those of Boggs & Chinault (2) based on a FISH assay in female lymphoblasts, they are the opposite of observations recently reported by Hansen et al. (3) using a different technique. Because our conclusions about the inactive X were deduced from the behavior of the active X in male cells, we reexamined the time when these loci replicate on the human inactive X chromosome isolated from its homolog in somatic cell hybrids. We also studied the same chromosome as an active X in related hybrids. The results provide direct evidence that the expressed XIST locus on the inactive X replicates earlier than its repressed homolog on the active X and earlier than the FRAXA locus which is repressed on this chromosome. The silent XIST locus on the active X replicates late along with F8C which is also not transcribed in these cells. Possible reasons for the different results obtained by Hansen et al. (3) are discussed.