EHEC O157 from A to T: EHEC O157:H7 epidemiology supplemented with long-read sequencing

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC O157) is a key zoonotic pathogen responsible for large food-borne outbreaks worldwide. Whole genome sequencing is a relatively novel technology being utilised by Public Health agencies to determine isolate relationship and inform outbreak investigations. However, the main implementation of whole genome sequencing currently utilises “short-read” sequencing which fails to obtain complete information on prophages and genome structure due to the presence of multiple repeat regions in EHEC O157 genomes. In collaboration with Public Health England, this research helped deploy short-read sequencing approaches for routine use at the Scottish Escherichia coli Reference Laboratory (SERL). This has allowed the SERL and affiliated Scottish epidemiologists to better determine whether isolates are related and trace the source of outbreaks. The “long-read” Pacific Biosciences (PacBio) sequencing platform was then used to analyse the complete prophage content (bacteriophage DNA integrated in the chromosome) of a subset of strains. Specifically, the analysis took an in-depth look at prophages encoding the main Shiga toxins (Stx) responsible for the serious pathology associated with EHEC O157 infections. In addition, the sequencing method allowed the observation of large chromosomal rearrangements (LCRs), potentially mediated by areas of homologies present in the prophage population. The significance of such LCRs is still being investigated but the genome plasticity may act to allow the bacterial strain to ‘switch’ phenotypes for niche adaptation. The potential of using “long-read” methods alongside routine “short-read” sequencing of EHEC O157 for public health benefit was investigated and its value demonstrated for outbreaks. For example, by enabling a more accurate prediction of the host/source attribution of an infection strain based on analysis of the Stx-encoding prophage within the isolate. While such approaches show considerable promise, costs and accuracy issues (depending on the platform, PacBio vs Oxford Nanopore MinIon) will need to be surmounted, and the underlying biology studied further, before their use could usurp more high throughput “short-read” methods

Assessment of The Geotechnical Properties of Municipal Solid Waste and Its Effect on The Surrounding Soil: A Review

Due to rapid growing of population and active lifestyle, massive amounts of municipal solid waste (MSW) are produced worldwide. The MSW can harm the environment and threaten the land if the dumping sites are not managed scientifically. The geotechnical properties of MSW are the key parameters required in the landfill operations and waste management facilities. Hence, presence of the geotechnical properties data of the waste can assist engineers in selecting possible solutions for extension of the landfill and obtaining prior background data for the evaluation and design of landfills. MSW disposal changes the geotechnical properties of soil. Also, alterations in the geotechnical properties of soils may contribute to the physical and physico-chemical interactions between soil and contaminants of the dumping sites. As leachate, which is generated by the waste, penetrates into the soil, it moves pollutants into the soil and influences the strength and stability of the soil. The main objective of this research is to summarize the most recent literature of the physical and mechanical properties of MSW, and their influence on the geotechnical properties of soil. The findings of numerous investigations on the physical and mechanical characteristics of MSW and soil influenced by MSW are presented and discussed. Depending on the reviewed research studies, it can be observed that the engineering characteristics of MSW are complicated and varied for various reasons. The waste components and degradation process can cause an increase in moisture content and unit weight, and a decrease in organic content, hydraulic conductivity and compressibility of MSW. Additionally, MSW sites significantly impact the physical and mechanical characteristics of underlain and surrounding soil and deteriorate the soil quality. Further, it was noticed that the influence of dumping on soil is reduced with depth due to less interaction between the soil and wast

Evolution of a zoonotic pathogen:investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing

Enterohaemorrhagic Escherichia coli (EHEC) O157 is a zoonotic pathogen for which colonization of cattle and virulence in humans is associated with multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of EHEC O157 continues to develop primarily based on core genome analyses; however, such short-read sequences have limited value for the analysis of prophage content and its chromosomal location. In this study, we applied Single Molecule Real Time (SMRT) sequencing, using the Pacific Biosciences long-read sequencing platform, to isolates selected from the main sub-clusters of this clonal group. Prophage regions were extracted from these sequences and from published reference strains. Genome position and prophage diversity were analysed along with genetic content. Prophages could be assigned to clusters, with smaller prophages generally exhibiting less diversity and preferential loss of structural genes. Prophages encoding Shiga toxin (Stx) 2a and Stx1a were the most diverse, and more variable compared to prophages encoding Stx2c, further supporting the hypothesis that Stx2c-prophage integration was ancestral to acquisition of other Stx types. The concept that phage type (PT) 21/28 (Stx2a+, Stx2c+) strains evolved from PT32 (Stx2c+) was supported by analysis of strains with excised Stx-encoding prophages. Insertion sequence elements were over-represented in prophage sequences compared to the rest of the genome, showing integration in key genes such as stx and an excisionase, the latter potentially acting to capture the bacteriophage into the genome. Prophage profiling should allow more accurate prediction of the pathogenic potential of isolates

SnapperDB: A database solution for routine sequencing analysis of bacterial isolates

Real-time surveillance of infectious disease using whole genome sequencing data poses challenges in both result generation and communication. SnapperDB represents a set of tools to store bacterial variant data and facilitate reproducible and scalable analysis of bacterial populations.  We also introduce the ‘SNP address’ nomenclature to describe the relationship between isolates in a population to the single nucleotide resolution. Summary:  We announce the release of SnapperDB v1.0 a program for scalable routine SNP analysis and storage of microbial populations. Availability:  SnapperDB is implemented as a python application under the open source BSD license.  All code and user guides are available at https://github.com/phe-bioinformatics/snapperdb. Reference genomes and SnapperDB configs are available at https://github.com/phe-bioinformatics/snapperdb_references Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.</p

Genome structural variation in Escherichia coli O157:H7

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments

Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium

Evolutionary Context of Non–Sorbitol-Fermenting Shiga Toxin–Producing Escherichia coli O55:H7

In July 2014, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a-encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway.

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity