Next generation sequencing analysis of nine Corynebacterium ulcerans isolates reveals zoonotic transmission and a novel putative diphtheria toxin-encoding pathogenicity island

Background: Toxigenic Corynebacterium ulcerans can cause a diphtheria-like illness in humans and have been found in domestic animals, which were suspected to serve as reservoirs for a zoonotic transmission. Additionally, toxigenic C. ulcerans were reported to take over the leading role in causing diphtheria in the last years in many industrialized countries. Methods: To gain deeper insights into the tox gene locus and to understand the transmission pathway in detail, we analyzed nine isolates derived from human patients and their domestic animals applying next generation sequencing and comparative genomics. Results: We provide molecular evidence for zoonotic transmission of C. ulcerans in four cases and demonstrate the superior resolution of next generation sequencing compared to multi-locus sequence typing for epidemiologic research. Additionally, we provide evidence that the virulence of C. ulcerans can change rapidly by acquisition of novel virulence genes. This mechanism is exemplified by an isolate which acquired a prophage not present in the corresponding isolate from the domestic animal. This prophage contains a putative novel virulence factor, which shares high identity with the RhuM virulence factor from Salmonella enterica but which is unknown in Corynebacteria so far. Furthermore, we identified a putative pathogenicity island for C. ulcerans bearing a diphtheria toxin gene. Conclusion: The novel putative diphtheria toxin pathogenicity island could provide a new and alternative pathway for Corynebacteria to acquire a functional diphtheria toxin-encoding gene by horizontal gene transfer, distinct from the previously well characterized phage infection model. The novel transmission pathway might explain the unexpectedly high number of toxigenic C. ulcerans

One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation

<div><h3>Background</h3><p>In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.</p> <h3>Methodology</h3><p>To address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.</p> <h3>Results and Conclusions</h3><p>We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.</p> </div

Development of Plasma Vitellogenin Assay for Estrogenic Effects of Endocrine-Disrupting Chemicals Using Ovariectomized Goldfish (Carassius auratus)

Plasma vitellogenin (VTG) assay was developed using ovariectomized goldfish (Carassius auratus) for determining the estrogenic effects of endocrine-disrupting chemicals. In a laboratory study, we assessed the estrogenic activity of commercial fish diets by using a diet for ornamental carp (CD) and a casein-based formulated fish diet (FD), which was shown to not contain soybean or fish meal in a previous study. In ovariectomized fish, plasma VTG concentrations were significantly higher in the CD-fed group than in the FD-fed group. These results indicate that the estrogen activity of CD may be high enough to cause induction of plasma VTG in ovariectomized goldfish as previously observed in male goldfish. Moreover, the effect of estrogen on plasma VTG induction was confirmed by significant plasma VTG production following the exposure of FD-fed ovariectomized goldfish to a nominal estradiol-17β concentration of 100 μg/l for 31 days. Our data suggest that induction of plasma VTG using ovariectomized goldfish is a good tool for evaluating the estrogenic effects of endocrine-disrupting chemicals