Heat survival of Clostridium difficile spores in ground meat during cooking process

Introduction: Clostridium difficile is a spore-forming pathogen considered as a major cause of enteric disease in humans, with faecal-oral route as the primary mode of transmission. However, recent studies have reported the occurrence of C. difficile in ground meats at retail stores, indicating that foods could be an additional source of infection in the community. Purpose: The objective of this study was to determine the resistance of C. difficile spores in contaminated ground meat during cooking process. Methods: Prior to testing, to obtain spores and to enhance heterogeneity, spores of two different strains were produced in two nutritious broths. C. difficile spores were experimentally inoculated in 45 g of ground meat (beef and pork) in order to obtain a final contamination of 4,500 ufc g-1. Six heating temperatures (70, 75, 80, 85, 90 and 95°C) were chosen. Samples were heating in a water bath with an integrated program for time-temperature. One sample without inoculum was used as control with a temperature probe placed inside. Once the desired temperature was research in the core of the sample, the heat treatment was prolonged for 10 min. Subsequently, all the samples were placed on the chilling room (4°C) before analyse. These experiments were conducted in duplicate with a spore enumeration in triplicate. Results: Heating contaminated ground meat at 70, 75 and 80°C for 10 min was not effective for C. difficile spores inhibition. However, 10 min of heat shock at 80°C was the only temperature that significantly reduced the number of countable colonies. Heat treatment at 85°C (or more) inhibits the germination of both of the strains tested. Significance: Ensure that ground meat, like burgers or sausages, is heated to more than 85°C would be an important measure to reduce the risk of C. difficile food transmission.ZooDif

Comparison and molecular characterization of animal and human Clostridium difficile strains

The main objective of this study was to characterize and compare animal and human C. difficile strains with respect to the PCR-ribotype and the antibiotic resistance. Multilocus sequence typing analysis (MLST) was performed in order to study clonal relationships of the isolates

Parameterized tests of the strong-field dynamics of general relativity using gravitational wave signals from coalescing binary black holes: Fast likelihood calculations and sensitivity of the method

Thanks to the recent discoveries of gravitational wave signals from binary black hole mergers by Advanced Laser Interferometer Gravitational Wave Observatory and Advanced Virgo, the genuinely strong-field dynamics of spacetime can now be probed, allowing for stringent tests of general relativity (GR). One set of tests consists of allowing for parametrized deformations away from GR in the template waveform models and then constraining the size of the deviations, as was done for the detected signals in previous work. In this paper, we construct reduced-order quadratures so as to speed up likelihood calculations for parameter estimation on future events. Next, we explicitly demonstrate the robustness of the parametrized tests by showing that they will correctly indicate consistency with GR if the theory is valid. We also check to what extent deviations from GR can be constrained as information from an increasing number of detections is combined. Finally, we evaluate the sensitivity of the method to possible violations of GR.Comment: 19 pages, many figures. Matches PRD versio

Clostridium difficile: an emerging zoonotic pathogen ?

Clostridium difficile is an anaerobic, spore-forming bacterium that remains the main cause of nosocomial diarrhoea in humans after use of antibiotics. C. difficile has also been described in other environments outside of hospitals, such as soil, river and seawater samples (Pasquale et al., 2011) and in animals, in which it can also cause enteric disease (Songer and Anderson, 2006). The possibility of transmission of C. difficile pathogenic isolates between animals, environments and humans has been suggested (Janezic et al., 2012). In recent years, the interest in C. difficile in food and in food animals has increased, leading to studying animals as a possible reservoir and a potential risk for food borne infections linked to C. difficile. Studies in various countries have determined differences in the prevalence of C. difficile in animals just before slaughter (Houser et al., 2012; Rodriguez et al., 2012) as well as in retail meat (Houser et al., 2012). In addition, many types, including PCR-ribotype 078, are present in humans, animals and meat (Janezic et al.; Weese et al., 2009). The objective of this study was to evaluate the presence of C. difficile at the slaughterhouse and in retail meat. Intestinal and carcass samples were collected from pigs and cattle at a single slaughterhouse. Raw meat (beef and pork) was obtained from the retail trade. C. difficile was isolated in 1% and 9.9% of the pig and cattle intestinal contents and in 7.9% and 7% of cattle and pig carcass samples respectively. From retail meat, C. difficile was recovered from 2.3% of the beef samples and from 4.7% of the pork samples. A total of 21 different PCR-ribotypes were identified with a large percentage of types 078 and 014. This study confirms that animals are carriers of C. difficile at slaughter, and that carcass contamination occurs inside the slaughterhouse. Furthermore the results obtained also reveal the presence of toxigenic C. difficile in retail meat in Belgium with a predominance of isolates correlated with the PCR-ribotypes involved in human C. difficile infections

Immunogenicity and protection efficacy of a naked self-replicating mRNA-based Zika virus vaccine

To combat emerging infectious diseases like Zika virus (ZIKV), synthetic messenger RNAs (mRNAs) encoding viral antigens are very attractive as they allow a rapid, generic, and flexible production of vaccines. In this work, we engineered a self-replicating mRNA (sr-mRNA) vaccine encoding the pre-membrane and envelope (prM-E) glycoproteins of ZIKV. Intradermal electroporation of as few as 1 µg of this mRNA-based ZIKV vaccine induced potent humoral and cellular immune responses in BALB/c and especially IFNAR1-/- C57BL/6 mice, resulting in a complete protection of the latter mice against ZIKV infection. In wild-type C57BL/6 mice, the vaccine resulted in very low seroconversion rates and antibody titers. The potency of the vaccine was inversely related to the dose of mRNA used in wild-type BALB/c or C57BL/6 mice, as robust type I interferon (IFN) response was determined in a reporter mice model (IFN-β+/Δβ-luc). We further investigated the inability of the sr-prM-E-mRNA ZIKV vaccine to raise antibodies in wild-type C57BL/6 mice and found indications that type I IFNs elicited by this naked sr-mRNA vaccine might directly impede the induction of a robust humoral response. Therefore, we assume that the efficacy of sr-mRNA vaccines after intradermal electroporation might be increased by strategies that temper their inherent innate immunogenicity

First isolation of Clostrioides difficile from smoked and dried freshwater fish in Cambodia

In Cambodia, freshwater aquaculture is the most important source of food production. Fresh fish meat is considered a highly perishable food that requires the use of different manipulations and preservation techniques to inhibit the proliferation of undesirable bacteria. These bacteria are naturally present in the raw product or could be acquired during manipulation by cross-contamination. Many studies worldwide have investigated the epidemiology of Clostridioides difficile (C. difficile) in food, but to date, there are no publications about the bacterium in ready-to-eat fish or descriptions in Cambodia. The objective of this study was to assess the presence of C. difficile in one of the main food supplies of this country, smoked freshwater fish, originating from different provinces. A total of 25 samples were collected directly from local markets, yielding 4 C. difficile isolates and an overall recovery rate of 16%. Most of the isolates were toxigenic and classified as rare PCR profiles, and they were resistant to clindamycin. These findings indicate contamination during handling and/or contamination of the raw fish, followed by insufficient heat treatment to kill the spores. The presence of C. difficile in smoked and dried fish implies a potential risk of human exposure, contamination and infection

Functional analysis of the Parkinson's disease associated genes, parkin and PINK1, in vitro

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Our understanding of the disease has been enhanced by the discovery of gene mutations in rare families with eariy-onset autosomal recessive juvenile parkinsonism (ARJP). The first gene to be identified was parkin and two further genes have been identified namely, DJ-1 and PINK1. The latter was discovered in our laboratory. The work of this thesis has aimed to increase our understanding of how these newly discovered genes play a role in neuronal survival and how disease-causing mutations result in neurodegenerarJon. Utilising gene transfer techniques and transient and stable cell culture expression systems, the function of parkin and PINK1 was investigated in human dopaminergic SH-SY5Y neuroblastoma cell lines. The work of this thesis has confirmed and extended previous reports that parkin over-expression confers neuroprotection against a variety of cellular stresses implicated in PD. Moreover, this work has showed for the first time that endogenous parkin protein can localize to aggregates known as "aggresomes' following stress and that the formation of these parkin positive aggresomes can be dissociated from parkin's effect on neuronal survival. Furthermore, this work describes the first functional characterization of PINK1. It demonstrates that PINK1 localizes to the mitochondria in neuronal cells where it may play a neuroprotective role against cellular stress. Moreover, this effect is abrogated by disease causing mutations in PINK1. This work also reports on the characterisation of novel PINK1 antibodies and shows for the first time that PINK1 can localize to aggresomes and the mechanism is linked to mitochondrial recruitment. The work of this thesis sheds light on the increasing importance of the ubiquitin proteasome system and mitochondria in the pathogenesis of PD. Improved understanding of these cellular processes should lead to more effective treatments for this devastating disease

A Systematic Review of the Efficacy and Toxicity of Brachytherapy Boost Combined with External Beam Radiotherapy for Nonmetastatic Prostate Cancer

CONTEXT: The optimum use of brachytherapy (BT) combined with external beam radiotherapy (EBRT) for localised/locally advanced prostate cancer (PCa) remains uncertain.OBJECTIVE: To perform a systematic review to determine the benefits and harms of EBRT-BT.EVIDENCE ACQUISITION: Ovid MEDLINE, Embase, and EBM Reviews-Cochrane Central Register of Controlled Trials databases were systematically searched for studies published between January 1, 2000 and June 7, 2022, according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Eligible studies compared low- or high-dose-rate EBRT-BT against EBRT ± androgen deprivation therapy (ADT) and/or radical prostatectomy (RP) ± postoperative radiotherapy (RP ± EBRT). The main outcomes were biochemical progression-free survival (bPFS), severe late genitourinary (GU)/gastrointestinal toxicity, metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival (OS), at/beyond 5 yr. Risk of bias was assessed and confounding assessment was performed. A meta-analysis was performed for randomised controlled trials (RCTs).EVIDENCE SYNTHESIS: Seventy-three studies were included (two RCTs, seven prospective studies, and 64 retrospective studies). Most studies included participants with intermediate-or high-risk PCa. Most studies, including both RCTs, used ADT with EBRT-BT. Generally, EBRT-BT was associated with improved bPFS compared with EBRT, but similar MFS, CSS, and OS. A meta-analysis of the two RCTs showed superior bPFS with EBRT-BT (estimated fixed-effect hazard ratio [HR] 0.54 [95% confidence interval {CI} 0.40-0.72], p &lt; 0.001), with absolute improvements in bPFS at 5-6 yr of 4.9-16%. However, no difference was seen for MFS (HR 0.84 [95% CI 0.53-1.28], p = 0.4) or OS (HR 0.87 [95% CI 0.63-1.19], p = 0.4). Fewer studies examined RP ± EBRT. There is an increased risk of severe late GU toxicity, especially with low-dose-rate EBRT-BT, with some evidence of increased prevalence of severe GU toxicity at 5-6 yr of 6.4-7% across the two RCTs.CONCLUSIONS: EBRT-BT can be considered for unfavourable intermediate/high-risk localised/locally advanced PCa in patients with good urinary function, although the strength of this recommendation based on the European Association of Urology guideline methodology is weak given that it is based on improvements in biochemical control.PATIENT SUMMARY: We found good evidence that radiotherapy combined with brachytherapy keeps prostate cancer controlled for longer, but it could lead to worse urinary side effects than radiotherapy without brachytherapy, and its impact on cancer spread and patient survival is less clear.</p

Identification of Z-Tyr-Ala-CHN 2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN 2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN 2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN 2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication