Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013

Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013

COPLA, a taxonomic classifier of plasmids

Background: Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. Results: We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. Conclusions: COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.This work was supported by the Spanish Ministry of Science and Innovation [PID2020-117923GB-I00 to FdlC]; the Spanish Ministry of Economy, Industry and Competitiveness [DI-17-09164 to SR-S]; and USA Centers for Disease Control and Prevention [200-2019-06679 to FdlC]. The funders had no role in the design of the study, nor in the collection, analysis, and interpretation of data, nor in writing the manuscript included in this submission

Carbapenem-Resistant Bacteria Recovered from Faeces of Dairy Cattle in the High Plains Region of the USA

OBJECTIVE:A study was conducted to recover carbapenem-resistant bacteria from the faeces of dairy cattle and identify the underlying genetic mechanisms associated with reduced phenotypic susceptibility to carbapenems. METHODS:One hundred and fifty-nine faecal samples from dairy cattle were screened for carbapenem-resistant bacteria. Phenotypic screening was conducted on two media containing ertapenem. The isolates from the screening step were characterised via disk diffusion, Modified Hodge, and Carba NP assays. Carbapenem-resistant bacteria and carbapenemase-producing isolates were subjected to Gram staining and biochemical testing to include Gram-negative bacilli. Whole genome sequencing was performed on bacteria that exhibited either a carbapenemase-producing phenotype or were not susceptible to ertapenem and were presumptively Enterobacteriaceae. RESULTS:Of 323 isolates collected from the screening media, 28 were selected for WGS; 21 of which were based on a carbapenemase-producing phenotype and 7 were presumptively Enterobacteriaceae and not susceptible to ertapenem. Based on analysis of WGS data, isolates included: 3 Escherichia coli harbouring blaCMY-2 and truncated ompF genes; 8 Aeromonas harbouring blacphA-like genes; 1 Acinetobacter baumannii harbouring a novel blaOXA gene (blaOXA-497); and 6 Pseudomonas with conserved domains of various carbapenemase-producing genes. CONCLUSIONS:Carbapenem resistant bacteria appear to be rare in cattle. Nonetheless, carbapenem-resistant bacteria were detected across various genera and were found to harbour a variety of mechanisms conferring reduced susceptibility. The development and dissemination of carbapenem-resistant bacteria in livestock would have grave implications for therapeutic treatment options in human medicine; thus, continued monitoring of carbapenem susceptibility among enteric bacteria of livestock is warranted

Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroup-specific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP_O26-E, which was previously identified only in stx 2-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces

Genome Sequences of 18 Salmonella enterica Serotype Hadar Strains Collected from Patients in the United States

Despite being linked to a number of recent poultry-associated outbreaks in the United States, few reference genomes are available for Salmonella enterica serotype Hadar. Here, we address this need by reporting 18 Salmonella Hadar genomes from samples collected from patients in the United States between 2014 and 2020

Challenges for IT-Enabled Formative Assessment of Complex 21st Century Skills

In this article, we identify and examine opportunities for formative assessment provided by information technologies (IT) and the challenges which these opportunities present. We address some of these challenges by examining key aspects of assessment processes that can be facilitated by IT: datafication of learning; feedback and scaffolding; peer assessment and peer feedback. We then consider how these processes may be applied in relation to the assessment of horizontal, general complex 21st century skills (21st CS), which are still proving challenging to incorporate into curricula as well as to assess. 21st CS such as creativity, complex problem solving, communication, collaboration and self-regulated learning contain complex constructs incorporating motivational and affective components. Our analysis has enabled us to make recommendations for policy, practice and further research. While there is currently much interest in and some progress towards the development of learning/assessment analytics for assessing 21st CS, the complexity of assessing such skills, together with the need to include affective aspects means that using IT-enabled techniques will need to be combined with more traditional methods of teacher assessment as well as peer assessment for some time to come. Therefore learners, teachers and school leaders must learn how to manage the greater variety of sorts and sources of feedback including resolving tensions of inconsistent feedback from different sources

Online collaboration and cooperation : the recurring importance of evidence, rationale and viability

This paper investigates collaboration in teaching and learning and draws out implications for the promotion of collaboration within online environments. It is divided into four sections. First the case for collaboration, including specifically cooperative approaches, is explored. This case revolves around the impact of collaboration on the quality of learning and on learning outcomes. Collaboration is seen as constrained by context but, if structured and rewarded, it will bring important motivational and cognitive benefits. Next, the case for online collaboration is examined. This is based on longstanding arguments about the benefits of working together albeit in an environment which offers greater reach; a mix of media; and archives of interaction. The third section of the paper compares perspectives on online collaboration with a longer tradition of research into collaboration in general; it critiques the idea that online mediation offers a paradigm change in teaching and learning. The fourth section of the paper considers future directions for promoting online collaboration

Pathways of gene dissemination within the Salmonella enterica plasmidome

Trabajo presentado en el International symposium Salmonella and salmonellosis, celebrado en Saint-Malo (Francia) del 20 al 22 de junio de 2022