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Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L −1 ). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant ( n  = 23), but not susceptible ( n  = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections. Cases of C. difficile (CD) resistant to metronidazole have been reported but the mechanism remains enigmatic. Here the authors identify a plasmid, which correlates with metronidazole resistance status in a large international collection of CD isolates, and demonstrate that the plasmid can confer metronidazole resistance.

Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrP Sc  accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8–9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrP Sc  accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie.

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.

The role of microRNAs (miRNAs) in neurodegenerative diseases has gained significant attention because of their involvement in gene regulation and potential as biomarkers. In prion diseases, including scrapie, miRNAs may modulate pathogenesis and disease progression. This study investigated circulating miRNA profiles in the blood of sheep naturally affected by scrapie at preclinical and clinical stages using small RNA sequencing and RT-qPCR validation. While only one novel miRNA was dysregulated in preclinical blood samples, 66 previously annotated miRNAs were significantly dysregulated in clinical sheep compared with healthy sheep. These miRNAs are associated with pathways commonly altered in neurodegenerative diseases, such as autophagy, ubiquitin-mediated proteolysis, and endoplasmic reticulum protein processing. Notably, miR-1271-5p, let-7f-5p, miR-186-5p, and miR-425-5p were consistently upregulated in the central nervous system of clinical animals, replicating the results observed in blood, with an increasing trend already in the preclinical stage and a strong correlation with neuropathological prion features. Additionally, predicted target genes such as UBQLN2 , PGK1 , KRAS , and CLTC were inversely expressed relative to these miRNAs, supporting their regulatory roles. These findings highlight the relevance of circulating miRNAs in prion neuropathology and support further research into the specific functional roles of these miRNAs and their predictive capacity for disease progression.

Classical scrapie is a prion disease that affects goats and sheep and is transmitted mainly horizontally. However, infection can also occur vertically, from mother to offspring, but the exact mechanism is unclear. The possibilities include transmission during pregnancy, birth, or after birth. Some studies have reported that prions are present in various reproductive tissues and fluids, suggesting that vertical transmission could occur through the germ line. However, the presence of prions in female reproductive tissues and oocytes has not been extensively studied, so their role in vertical transmission is unknown. Oocytes and ovarian tissues from 25 naturally infected ewes (ARQ/ARQ and VRQ/VRQ genotypes) and healthy controls were analysed. The samples were subjected to PMCA for prion detection. Histopathological and immunohistochemical evaluations of ovarian tissues were also performed. We demonstrated that PrP Sc can accumulate in the oocytes and ovaries of scrapie-infected ewes, indicating a potential germline route of transmission. Prion accumulation appears to be influenced by the host genotype and prion strain, emphasizing the need for ultrasensitive detection techniques. Further research under controlled conditions is necessary to elucidate the mechanisms and implications for disease control and breeding programs.

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrPC to its abnormal and misfolded isoform PrPSc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrPSc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrPSc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the UbG76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the UbG76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases.

Chronic accumulation of misfolded proteins such as PrP Sc can alter the endoplasmic reticulum homeostasis triggering the unfolded protein response (UPR). In this pathogenic event, the molecular chaperones play an important role. Several reports in humans and animals have suggested that neurodegeneration is related to endoplasmic reticulum stress in diseases caused by the accumulation of misfolded proteins. In this study, we investigated the expression of three endoplasmic reticulum stress markers: PERK (protein kinase R-like endoplasmic reticulum kinase), BiP (binding immunoglobulin protein), and PDI (Protein Disulfide Isomerase). In addition, we evaluated the accumulation of ubiquitin as a marker for protein degradation mediated by the proteasome. These proteins were studied in brain tissues of sheep affected by scrapie in clinical and preclinical stages of the disease. Results were compared with those observed in healthy controls. Scrapie-infected sheep showed significant higher levels of PERK, BiP/Grp78 and PDI than healthy animals. As we observed before in models of spontaneous prion disease, PDI was the most altered ER stress marker between scrapie-infected and healthy sheep. Significantly increased intraneuronal and neuropil ubiquitinated deposits were observed in certain brain areas in scrapie-affected animals compared to controls. Our results suggest that the neuropathological and neuroinflammatory phenomena that develop in prion diseases cause endoplasmic reticulum stress in brain cells triggering the UPR. In addition, the significantly higher accumulation of ubiquitin aggregates in scrapie-affected animals suggests an impairment of the ubiquitin-proteasome system in natural scrapie. Therefore, these proteins may contribute as biomarkers and/or therapeutic targets for prion diseases.

Autophagy is a dynamic cellular mechanism involved in protein and organelle turnover through lysosomal degradation. Autophagy regulation modulates the pathologies associated with many neurodegenerative diseases. Using sheep naturally infected with scrapie as a natural animal model of prion diseases, we investigated the regulation of autophagy in the central nervous system (CNS) during the clinical phase of the disease. We present a gene expression and protein distribution analysis of different autophagy-related markers and investigate their relationship with prion-associated lesions in several areas of the CNS. Gene expression of autophagy markers ATG5 and ATG9 was downregulated in some areas of scrapie brains. In contrast, ATG5 protein accumulates in medulla oblongata and positively correlates with prion deposition and scrapie-related lesions. The accumulation of this protein and p62, a marker of autophagy impairment, suggests that autophagy is decreased in the late phases of the disease. However, the increment of LC3 proteins and the mild expression of p62 in basal ganglia and cerebellum, primarily in Purkinje cells, suggests that autophagy machinery is still intact in less affected areas. We hypothesize that specific cell populations of the CNS may display neuroprotective mechanisms against prion-induced toxicity through the induction of PrP Sc clearance by autophagy.

Background Chronic wasting disease (CWD) is a prion disease affecting members of the Cervidae family. PrP C primary structures play a key role in CWD susceptibility resulting in extended incubation periods and regulating the propagation of CWD strains. We analyzed the distribution of abnormal prion protein (PrP CWD ) aggregates in brain and peripheral organs from orally inoculated white-tailed deer expressing four different PRNP genotypes: Q95G96/Q95G96 (wt/wt), S96/wt, H95/wt and H95/S96 to determine if there are substantial differences in the deposition pattern of PrP CWD between different PRNP genotypes. Results Although we detected differences in certain brain areas, globally, the different genotypes showed similar PrP CWD deposition patterns in the brain. However, we found that clinically affected deer expressing H95 PrP C , despite having the longest survival periods, presented less PrP CWD immunoreactivity in particular peripheral organs. In addition, no PrP CWD was detected in skeletal muscle of any of the deer. Conclusions Our data suggest that expression of H95-PrP C limits peripheral accumulation of PrP CWD as detected by immunohistochemistry. Conversely, infected S96/wt and wt/wt deer presented with similar PrP CWD peripheral distribution at terminal stage of disease, suggesting that the S96-PrP C allele, although delaying CWD progression, does not completely limit the peripheral accumulation of the infectious agent.

This review aims to provide a comprehensive overview of the significant Clostridioides difficile molecular typing techniques currently employed in research and medical communities. The main objectives of this review are to describe the key molecular typing methods utilized in C. difficile studies and to highlight the epidemiological characteristics of the most prevalent strains on a global scale. Geographically distinct regions exhibit distinct strain types of C. difficile, with notable concordance observed among various typing methodologies. The advantages that next-generation sequencing (NGS) offers has changed epidemiology research, enabling high-resolution genomic analyses of this pathogen. NGS platforms offer an unprecedented opportunity to explore the genetic intricacies and evolutionary trajectories of C. difficile strains. It is relevant to acknowledge that novel routes of transmission are continually being unveiled and warrant further investigation, particularly in the context of zoonotic implications and environmental contamination.