1,721,036 research outputs found
Real-time PCR detection of the parasitic ciliate Ichthyophthirius multifiliis in water samples
No full textStaphylococcus lentus strain X11 16S ribosomal RNA gene, partial sequence
No full textStaphylococcus lentus strain X11 16S ribosomal RNA gene, partial sequence
GenBank: EF424601.
MetaMLST: multi-locus strain-level bacterial typing from metagenomic samples
Full text linkMetagenomic characterization of microbial communities has the potential to become a tool to identify pathogens in human samples. However, software tools able to extract strain-level typing information from metagenomic data are needed. Low-throughput molecular typing schema such as Multilocus Sequence Typing (MLST) are still widely used and provide a wealth of strain-level information that is currently not exploited by metagenomic methods. We introduce MetaMLST, a software tool that reconstructs the MLST loci of microorganisms present in microbial communities from metagenomic data. Tested on synthetic and spiked-in real metagenomes, the pipeline was able to reconstruct the MLST sequences with >98.5% accuracy at coverages as low as 1X. On real samples, the pipeline showed higher sensitivity than assembly-based approaches and it proved successful in identifying strains in epidemic outbreaks as well as in intestinal, skin and gastrointestinal microbiome samples
Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR
No full textThe main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multitihis. We developed a real-time PCR assay using SYBR Green (TM) intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of L multitihis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the L multifillis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R-2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 L multifiliis cells 1(-1). The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture
Accurate flow cytometric monitoring of Escherichia coli subpopulations on solid food treated with high pressure carbon dioxide
No full textAims: Evaluation of flow cytometry coupled with viability markers to monitor the inactivation of Escherichia coli cells spiked on solid food following High Pressure Carbon Dioxide (HPCD), a mild processing technology. Methods and Results: Flow cytometry (FCM) coupled with SYBR-Green I and Propidium Iodide was applied to monitor the effect of HPCD treatment on E. coli cells spiked on fresh cut carrots, therefore mimicking contamination of food products by faecal coliforms. FCM allowed to distinguish E. coli cells from carrot debris and natural flora, to evaluate the reduction of total cells, and to quantify viable and dead cells based on their membrane integrity after HPCD treatment. The comparison of FCM results with conventional cultivation methods revealed that HPCD treatments performed at 120 bar, 22 or 35°C for 5 min disrupted 43 and 53% of bacterial cells, respectively, and produced a large percentage of partially permeabilized (96·5 and 98%) cells. Conclusions: Although treatments at 22°C for 10 min and at 35°C for 7 min were sufficient to inhibit the ability of all E. coli cells to replicate with an inactivation of 8 Log, FCM analysis showed that the inactivation of intact cells was only 2-2·5 Log. A fraction of HPCD-treated cells maintained their metabolic activity and re-growth capacity, indicating that the treatment induces a transitory Viable But Not Cultivable (VNBC) state. Significance and Impact of the Study: Under stress conditions many pathogens enter in a VBNC state, thus escaping detection by traditional cultivation methods. We provide the first evaluation of HPCD-mediated bacterial inactivation on fresh food using FCM coupled with viability markers, which should assist in the prevention of food-associated health risks. © 2014 The Society for Applied Microbiology
Staphylococcus kloosii strain X79 16S ribosomal RNA gene, partial sequence
No full textStaphylococcus kloosii strain X79 16S ribosomal RNA gene, partial sequence
GenBank: EF424600.
Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis
No full textAn expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process
Insights into the genome structure of four acetogenic bacteria with specific reference to the Wood–Ljungdahl pathway
No full textAcetogenic bacteria are obligate anaerobes with the ability of converting carbon dioxide and other one-carbon substrates into acetate through the Wood–Ljungdahl (WL) pathway. These substrates are becoming increasingly important feedstock in industrial microbiology. The main potential industrial application of acetogenic bacteria is the production of metabolites that constitute renewable energy sources (biofuel); such bacteria are of particular interest for this purpose thanks to their low energy requirements for large-scale cultivation. Here, we report new genome sequences for four species, three of them are reported for the first time, namely Acetobacterium paludosum DSM 8237, Acetobacterium tundrae DSM 917, Acetobacterium bakii DSM 8239, and Alkalibaculum bacchi DSM 221123. We performed a comparative genomic analysis focused on the WL pathway's genes and their encoded proteins, using Acetobacterium woodii as a reference genome. The Average Nucleotide Identity (ANI) values ranged from 70% to 95% over an alignment length of 5.4–6.5 Mbp. The core genome consisted of 363 genes, whereas the number of unique genes in a single genome ranged from 486 in A. tundrae to 2360 in A.bacchi. No significant rearrangements were detected in the gene order for the Wood–Ljungdahl pathway however, two species showed variations in genes involved in formate metabolism: A. paludosum harbor two copies of fhs1, and A. bakii a truncated fdhF1. The analysis of protein networks highlighted the expansion of protein orthologues in A. woodii compared to A. bacchi, whereas protein networks involved in the WL pathway were more conserved. This study has increased our understanding on the evolution of the WL pathway in acetogenic bacteria
Application of culture-independent methods for monitoring Listeria monocytogenes inactivation on food products
No full textWhen new food processing technologies are investigated as alternative to traditional thermal pasteurization processes, conventional cultivation-based methods are usually applied to evaluate microbial concentration before and after the treatment to determine the process efficiency. However, these standard methods lead to a typical underestimation of the microbes present in the sample, which may represent an issue when pathogenic strains have to be detected. Here, the efficiency of SC-CO2 pasteurization treatment in the inactivation of Listeria monocytogenes spiked on cured ham skin surface was evaluated using plate counts, flow cytometry (FCM) coupled with SYBR-Green I (SYBR-I) and propidium iodide (PI), and propidium monoazide quantitative PCR (PMA-qPCR), at different process conditions. SC-CO2 best performed at 12 MPa, 45 and 50 °C, resulting in a 7.5 log reduction of cultivable cells quantified by plate counts after 15 min of treatment, while FCM and PMA-qPCR revealed a 4 log and 2 log reduction of intact cells, respectively. This striking difference between culture-based and culture-independent quantification methods was independent from treatment time and indicated that a large fraction of the cells lost cultivability after treatment but maintained an intact membrane, likely entering in a so-called Viable But Not Culturable (VBNC) state. Our study highlights the usefulness of FCM and PMA-qPCR to assess the viability status of microbial populations and support their application in microbiological quality control in the food industry, in particular when mild pasteurization technologies are used
Staphylococcus epidermidis strain X70 16S ribosomal RNA gene, partial sequence.
No full textStaphylococcus epidermidis strain X70 16S ribosomal RNA gene, partial sequence. GenBank: EF424588.
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