Nicholas Maitland Thomson

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organizations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1565 positive samples (172 per 100 000 population) from 1376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6 % of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. In total, 1035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically distinct UK lineages were detected demonstrating local evolution, at a rate of ~2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with six care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients, indicating infection control measures were effective; and found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves.

Chickens and guinea fowl are commonly reared in Gambian homes as affordable sources of protein. Using standard microbiological techniques, we obtained 68 caecal isolates of Escherichia coli from 10 chickens and 9 guinea fowl in rural Gambia. After Illumina whole-genome sequencing, 28 sequence types were detected in the isolates (4 of them novel), of which ST155 was the most common (22/68, 32 %). These strains span four of the eight main phylogroups of E. coli, with phylogroups B1 and A being most prevalent. Nearly a third of the isolates harboured at least one antimicrobial resistance gene, while most of the ST155 isolates (14/22, 64 %) encoded resistance to ≥3 classes of clinically relevant antibiotics, as well as putative virulence factors, suggesting pathogenic potential in humans. Furthermore, hierarchical clustering revealed that several Gambian poultry strains were closely related to isolates from humans. Although the ST155 lineage is common in poultry from Africa and South America, the Gambian ST155 isolates belong to a unique cgMLST cluster comprising closely related (38–39 alleles differences) isolates from poultry and livestock from sub-Saharan Africa – suggesting that strains can be exchanged between poultry and livestock in this setting. Continued surveillance of E. coli and other potential pathogens in rural backyard poultry from sub-Saharan Africa is warranted.

Recombineering using bacteriophage lambda Red recombinase (λ-Red) uses homologous recombination to manipulate bacterial genomes and is commonly applied to disrupt genes to elucidate their function. This is often followed by the introduction of a wild-type copy of the gene on a plasmid to complement its function. This is often not, however, at a native copy number and the introduction of a chromosomal version of a gene can be a desirable solution to provide wild-type copy expression levels of an allele in trans. Here, we present a simple methodology based on the λ-Red-based ‘gene doctoring’ technique, where we developed tools used for chromosomal tagging in a conserved locus downstream of glmS and found no impact on a variety of important phenotypes. The tools described provide an easy, quick and inexpensive method of chromosomal modification for the creation of a library of insertion mutants to study gene function.

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3,200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organisations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1,565 positive samples (172 per 100,000 population) from 1,376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6% of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. 1,035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically-distinct UK lineages were detected demonstrating local evolution, at a rate of ∼2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a discrete sublineage associated with 6 care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients indicating infection control measures were effective; found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves. Major points In Norfolk and surrounding regions: 100 distinct UK lineages were identified. 16 UK lineages found in key workers were not observed in patients or in community care. 172 genomes from SARS-CoV-2 positive samples sequenced per 100,000 population representing 42.6% of all positive cases. SARS-CoV-2 genomes from 1035 cases sequenced to a high quality. Only 5 countries, out of 103, have sequenced more SARS-CoV-2 genomes than have been sequenced in Norfolk for this paper. Samples covered the entire first wave, March to August 2020. Stable evolutionary rate of 2 SNPs per month. D614G mutation is the dominant genotype and associated with increased transmission. No evidence of reinfection in 42 cases with longitudinal samples. WGS identified a sublineage associated with care facilities. WGS ruled out nosocomial outbreaks. Rapid WGS confirmed the relatedness of cases from an outbreak at a food processing facility.

Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between host species. We have investigated genomic diversity and antimicrobial resistance in Escherichia coli isolates from four species of non-human primates in the Gambia: Papio papio (n=22), Chlorocebus sabaeus (n=14), Piliocolobus badius (n=6) and Erythrocebus patas (n=1). We performed Illumina whole-genome sequencing on 101 isolates from 43 stools, followed by nanopore long-read sequencing on 11 isolates. We identified 43 sequence types (STs) by the Achtman scheme (ten of which are novel), spanning five of the eight known phylogroups of E. coli . The majority of simian isolates belong to phylogroup B2 – characterized by strains that cause human extraintestinal infections – and encode factors associated with extraintestinal disease. A subset of the B2 strains (ST73, ST681 and ST127) carry the pks genomic island, which encodes colibactin, a genotoxin associated with colorectal cancer. We found little antimicrobial resistance and only one example of multi-drug resistance among the simian isolates. Hierarchical clustering showed that simian isolates from ST442 and ST349 are closely related to isolates recovered from human clinical cases (differences in 50 and 7 alleles, respectively), suggesting recent exchange between the two host species. Conversely, simian isolates from ST73, ST681 and ST127 were distinct from human isolates, while five simian isolates belong to unique core-genome ST complexes – indicating novel diversity specific to the primate niche. Our results are of planetary health importance, considering the increasing contact between humans and wild non-human primates.

Chickens and guinea fowl are commonly reared in Gambian homes as affordable sources of protein. Using standard microbiological techniques, we obtained 68 caecal isolates of Escherichia coli from ten chickens and nine guinea fowl in rural Gambia. After Illumina whole-genome sequencing, 28 sequence types were detected in the isolates (four of them novel), of which ST155 was the most common (22/68, 32%). These strains span four of the eight main phylogroups of E. coli , with phylogroups B1 and A being most prevalent. Nearly a third of the isolates harboured at least one antimicrobial resistance gene, while most of the ST155 isolates (14/22, 64%) encoded resistance to ≥3 classes of clinically relevant antibiotics, as well as putative virulence factors, suggesting pathogenic potential in humans. Furthermore, hierarchical clustering revealed that several Gambian poultry strains were closely related to isolates from humans. Although the ST155 lineage is common in poultry from Africa and South America, the Gambian ST155 isolates belong to a unique cgMLST cluster comprised of closely related (38-39 alleles differences) isolates from poultry and livestock from sub-Saharan Africa—suggesting that strains can be exchanged between poultry and livestock in this setting. Continued surveillance of E. coli and other potential pathogens in rural backyard poultry from sub-Saharan Africa is warranted. Author notes: All supporting data and protocols have been provided within the article or as supplementary data files. Eleven supplementary figures and eight supplementary files are available with the online version of this article. Data summary: The genomic assemblies for the isolates reported here are available for download from EnteroBase ( http://enterobase.warwick.ac.uk/species/index/ecoli ) and the EnteroBase assembly barcodes are provided in File S2. Sequences have been deposited in the NCBI SRA, under the BioProject ID: PRJNA616250 and accession numbers SAMN14485281 to SAMN14485348 (File S2). Assemblies have been deposited in GenBank under the BioProject ID: PRJNA616250 and accession numbers CP053258 and CP053259 . Impact statement: Domestic birds play a crucial role in human society, in particular contributing to food security in low-income countries. Many households in Sub-Saharan Africa rear free-range chickens and guinea fowl, which are often left to scavenge for feed in and around the family compound, where they are frequently exposed to humans, other animals and the environment. Such proximity between backyard poultry and humans is likely to facilitate transmission of pathogens such as Escherichia coli or antimicrobial resistance between the two host species. Little is known about the population structure of E. coli in rural chickens and guinea fowl, although this information is needed to contextualise the potential risks of transmission of bacterial strains between humans and rural backyard poultry. Thus, we sought to investigate the genomic diversity of E. coli in backyard poultry from rural Gambia.

Among long-stay critically ill patients in the adult intensive care unit (ICU), there are often marked changes in the complexity of the gut microbiota. However, it remains unclear whether such patients might benefit from enhanced surveillance or from interventions targeting the gut microbiota or the pathogens therein. We therefore undertook a prospective observational study of 24 ICU patients, in which serial faecal samples were subjected to shotgun metagenomic sequencing, phylogenetic profiling and microbial genome analyses. Two-thirds of the patients experienced a marked drop in gut microbial diversity (to an inverse Simpson’s index of Enterococcus faecium . In some patients, we also saw an increase in the relative abundance of apparent commensal organisms in the gut microbiome, including the archaeal species Methanobrevibacter smithii . In conclusion, we have documented a dramatic absence of microbial diversity and pathogen domination of the gut microbiota in a high proportion of critically ill patients using shotgun metagenomics.

Flagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially restored using the tightly regulated rhamnose promoter, but wild-type motility was achieved with the T5 promoter, which, although leaky, allowed titration of induction strength. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.