Cancer invasion and anaerobic bacteria: new insights into mechanisms.

There is growing evidence that altered microbiota abundance of a range of specific anaerobic bacteria are associated with cancer, including Peptoniphilus spp., Porphyromonas spp., Fusobacterium spp., Fenollaria spp., Prevotella spp., Sneathia spp., Veillonella spp. and Anaerococcus spp. linked to multiple cancer types. In this review we explore these pathogenic associations. The mechanisms by which bacteria are known or predicted to interact with human cells are reviewed and we present an overview of the interlinked mechanisms and hypotheses of how multiple intracellular anaerobic bacterial pathogens may act together to cause host cell and tissue microenvironment changes associated with carcinogenesis and cancer cell invasion. These include combined effects on changes in cell signalling, DNA damage, cellular metabolism and immune evasion. Strategies for early detection and eradication of anaerobic cancer-associated bacterial pathogens that may prevent cancer progression are proposed.

A One Health Perspective on Salmonella enterica Serovar Infantis, an Emerging Human Multidrug-Resistant Pathogen.

Salmonella enterica serovar Infantis presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR). We analyzed whole-genome sequences of 5,284 Salmonella Infantis strains from 74 countries, isolated during 1989-2020 from a wide variety of human, animal, and food sources, to compare genetic phylogeny, AMR determinants, and plasmid presence. The global Salmonella Infantis population structure diverged into 3 clusters: a North American cluster, a European cluster, and a global cluster. The levels of AMR varied by Salmonella Infantis cluster and by isolation source; 73% of poultry isolates were multidrug resistant, compared with 35% of human isolates. This finding correlated with the presence of the pESI megaplasmid; 71% of poultry isolates contained pESI, compared with 32% of human isolates. This study provides key information for public health teams engaged in reducing the spread of this pathogen.

A decade of Campylobacter and Campylobacter bacteraemias in a district general hospital and the surrounding London and South East region, England.

BACKGROUND: Campylobacter bacteraemia is a rare complication of the most common bacterial gastrointestinal infection but is associated with significant morbidity and mortality. There is limited data describing current trends in surveillance and antimicrobial resistance for the Campylobacter strains involved. At the Epsom and St Helier's University Hospital (ESTH), we noted a marked increase in Campylobacter bacteraemia infections in 2021. METHODS: We extracted Campylobacter reports using the UK Health Security Agency's (UKHSA) Second Generation Surveillance System (laboratory reporting system) between 1st January 2012 and 31st December 2021. We reviewed patient records of patients with Campylobacter bacteraemia for details including presentation, past medical history, duration of hospital stay, and antibiotic use. RESULTS: Between 2012 and 2021, ESTH reported a total of 34 cases of Campylobacter bacteraemia. In 2021, the estimated incidence was 6.8 cases per 100,000 population and in the surrounding area, the incidence was 0.4 per 100,000 population. The incidence rate of Campylobacter bacteraemia in London and the South East region was significantly lower than ESTH (RR = 0.17, p < 0.0001). Campylobacter bacteraemia cases at ESTH reported a high number of co-morbidities (average number of comorbidities = 2.3) and had a duration of stay in hospital of a median of 7 days (IQR = 4-10 days). Campylobacter jejuni was the most commonly reported species for stool and blood Campylobacter in ESTH, London, and South East England. CONCLUSION: Campylobacter bacteraemia reports at ESTH were significantly (p < 0.001) higher than the surrounding London and South East region. While no common cause for the exceedance of Campylobacter bacteraemia has been identified, common risk factors for Campylobacter bacteraemia infection include underlying health conditions, being older, and male.

Characterisation of neonatal Staphylococcus capitis NRCS-A isolates compared with non NRCS-A Staphylococcus capitis from neonates and adults.

Staphylococcus capitis is a frequent cause of late-onset sepsis in neonates admitted to Neonatal Intensive Care Units (NICU). One clone of S. capitis, NRCS-A has been isolated from NICUs globally although the reasons for the global success of this clone are not well understood.We analysed a collection of S. capitis colonising babies admitted to two NICUs, one in the UK and one in Germany as well as corresponding pathological clinical isolates. Genome analysis identified a population structure of three groups; non-NRCS-A isolates, NRCS-A isolates, and a group of 'proto NRCS-A' - isolates closely related to NRCS-A but not associated with neonatal infection. All bloodstream isolates belonged to the NRCS-A group and were indistinguishable from strains carried on the skin or in the gut. NRCS-A isolates showed increased tolerance to chlorhexidine and antibiotics relative to the other S. capitis as well as enhanced ability to grow at higher pH values. Analysis of the pangenome of 138 isolates identified characteristic nsr and tarJ genes in both the NRCS-A and proto groups. A CRISPR-cas system was only seen in NRCS-A isolates which also showed enrichment of genes for metal acquisition and transport.We found evidence for transmission of S. capitis NRCS-A within NICU, with related isolates shared between babies and multiple acquisitions by some babies. Our data show NRCS-A strains commonly colonise uninfected babies in NICU representing a potential reservoir for potential infection. This work provides more evidence that adaptation to survive in the gut and on skin facilitates spread of NRCS-A, and that metal acquisition and tolerance may be important to the biology of NRCS-A. Understanding how NRCS-A survives in NICUs can help develop infection control procedures against this clone.

A global genomic analysis of Salmonella Concord reveals lineages with high antimicrobial resistance in Ethiopia.

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance.

Genome-wide analysis of genes involved in efflux function and regulation within Escherichia coli and Salmonella enterica serovar Typhimurium.

The incidence of multidrug-resistant bacteria is increasing globally, with efflux pumps being a fundamental platform limiting drug access and synergizing with other mechanisms of resistance. Increased expression of efflux pumps is a key feature of most cells that are resistant to multiple antibiotics. Whilst expression of efflux genes can confer benefits, production of complex efflux systems is energetically costly and the expression of efflux is highly regulated, with cells balancing benefits against costs. This study used TraDIS-Xpress, a genome-wide transposon mutagenesis technology, to identify genes in Escherichia coli and Salmonella Typhimurium involved in drug efflux and its regulation. We exposed mutant libraries to the canonical efflux substrate acriflavine in the presence and absence of the efflux inhibitor phenylalanine-arginine ß-naphthylamide. Comparisons between conditions identified efflux-specific and drug-specific responses. Known efflux-associated genes were easily identified, including acrAB, tolC, marRA, ramRA and soxRS, confirming the specificity of the response. Further genes encoding cell envelope maintenance enzymes and products involved with stringent response activation, DNA housekeeping, respiration and glutathione biosynthesis were also identified as affecting efflux activity in both species. This demonstrates the deep relationship between efflux regulation and other cellular regulatory networks. We identified a conserved set of pathways crucial for efflux activity in these experimental conditions, which expands the list of genes known to impact on efflux efficacy. Responses in both species were similar and we propose that these common results represent a core set of genes likely to be relevant to efflux control across the Enterobacteriaceae.

Determination and quantification of microbial communities and antimicrobial resistance on food through host DNA-depleted metagenomics.

Food products carry bacteria unless specifically sterilised. These bacteria can be pathogenic, commensal or associated with food spoilage, and may also be resistant to antimicrobials. Current methods for detecting bacteria on food rely on culturing for specific bacteria, a time-consuming process, or 16S rRNA metabarcoding that can identify different taxa but not their genetic content. Directly sequencing metagenomes of food is inefficient as its own DNA vastly outnumbers the bacterial DNA present. We optimised host DNA depletion enabling efficient sequencing of food microbiota, thereby increasing the proportion of non-host DNA sequenced 13-fold (mean; range: 1.3-40-fold) compared to untreated samples. The method performed best on chicken, pork and leafy green samples which had high mean prokaryotic read proportions post-depletion (0.64, 0.74 and 0.74, respectively), with lower mean prokaryotic read proportions in salmon (0.50) and prawn samples (0.19). We show that bacterial compositions and concentrations of antimicrobial resistance (AMR) genes differed by food type, and that salmon metagenomes were influenced by the production/harvesting method. The approach described in this study is an efficient and effective method of identifying and quantifying the predominant bacteria and AMR genes on food.

Comparative genomics of Campylobacter jejuni from clinical campylobacteriosis stool specimens.

BACKGROUND: Campylobacter jejuni is a pervasive pathogen of major public health concern with a complex ecology requiring accurate and informative approaches to define pathogen diversity during outbreak investigations. Source attribution analysis may be confounded if the genetic diversity of a C. jejuni population is not adequately captured in a single specimen. The aim of this study was to determine the genomic diversity of C. jejuni within individual stool specimens from four campylobacteriosis patients. Direct plating and pre-culture filtration of one stool specimen per patient was used to culture multiple isolates per stool specimen. Whole genome sequencing and pangenome level analysis were used to investigate genomic diversity of C. jejuni within a patient. RESULTS: A total 92 C. jejuni isolates were recovered from four patients presenting with gastroenteritis. The number of isolates ranged from 13 to 30 per patient stool. Three patients yielded a single C. jejuni multilocus sequence type: ST-21 (n = 26, patient 4), ST-61 (n = 30, patient 1) and ST-2066 (n = 23, patient 2). Patient 3 was infected with two different sequence types [ST-51 (n = 12) and ST-354 (n = 1)]. Isolates belonging to the same sequence type from the same patient specimen shared 12-43 core non-recombinant SNPs and 0-20 frameshifts with each other, and the pangenomes of each sequence type consisted of 1406-1491 core genes and 231-264 accessory genes. However, neither the mutation nor the accessory genes were connected to a specific functional gene category. CONCLUSIONS: Our findings show that the C. jejuni population recovered from an individual patient's stool are genetically diverse even within the same ST and may have shared common ancestors before specimens were obtained. The population is unlikely to have evolved from a single isolate at the time point of initial patient infection, leading us to conclude that patients were likely infected with a heterogeneous C. jejuni population. The diversity of the C. jejuni population found within individual stool specimens can inform future methodological approaches to attribution and outbreak investigations.

Impact of Salmonella genome rearrangement on gene expression.

In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure (GS) around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to GS identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved toward or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide-level measures of variation.

Use of Vitex doniana (black plum) and Abutilon hirtum (Florida keys) extracts as an integral part of phytomedicine in tackling multidrug-resistant Salmonella.

INTRODUCTION: The high prevalence and global spread of antibiotic resistance is driving the search for new antibacterial agents. Screening small molecules against specific bacterial targets has not yielded new compounds therefore functional assays and phenotypic screens are now being used. In Nigeria, drug resistance towards Salmonella is a major public health concern. METHODOLOGY: Nine fully characterized clinical Salmonella isolates, from the Department of Medical Microbiology, Jos University Teaching Hospital, Plateau State, Nigeria, were screened by broth microdilution for susceptibility to fractionated ethanol extracts of Vitex doniana and Abutilon hirtum. This was compared to the control organism ATCC25922 and a range antibiotics: CH (chloramphenicol), SP (sparfloxacin), AM (amoxicillin), CN (gentamicin), S (streptomycin), PEF (pefloxacin). RESULTS: The most common resistance profile was AM,CN,S with most isolates susceptible to fluoroquinolones. Activity was detected from both plant extracts with MICs of extracted fractions ranging from 150 - 300 µg/mL. Interestingly both plants produced extracts with bactericidal activity from 300 - 600 µg/mL. V. doniana exhibited better activity against the resistant Salmonella strains in terms of greater inhibition zones, but A. hirtum extracts were more consistently active against all isolates. In comparison with the synthetic drugs, both plant extracts exhibited activity against more isolates - this activity was bactericidal. CONCLUSIONS: Nigeria needs better anti-salmonella products and these results represent a starting point for antibiotic drug discovery.

Genetic characterization of Salmonella Infantis from South Africa, 2004-2016.

Salmonella Infantis is presenting an increasing risk to public health. Of particular concern are the reports of pESI, a multidrug resistance (MDR) encoding megaplasmid, in isolates from multiple countries, but little is known about its presence or diversity in South Africa. Whole genome sequences of 387 S. Infantis isolates from South Africa (2004-2020) were analysed for genetic phylogeny, recombination frequency, antimicrobial resistance (AMR) determinants, plasmid presence and overall gene content. The population structure of South African S. Infantis was substantially different to S. Infantis reported elsewhere; only two thirds of isolates belonged to eBG31, while the remainder were identified as eBG297, a much rarer group globally. Significantly higher levels of recombination were observed in the eBG297 isolates, which was associated with the presence of prophages. The majority of isolates were putatively susceptible to antimicrobials (335/387) and lacked any plasmids (311/387); the megaplasmid pESI was present in just one isolate. A larger proportion of eBG31 isolates, 19% (49/263), contained at least one AMR determinant, compared to eBG297 at 2% (3/124). Comparison of the pan-genomes of isolates from either eBG identified 943 genes significantly associated with eBG, with 43 found exclusively in eBG31 isolates and 34 in eBG297 isolates. This, along with the single nucleotide polymorphism distance and difference in resistance profiles, suggests that eBG31 and eBG297 isolates occupy different niches within South Africa. If antibiotic-resistant S. Infantis emerges in South Africa, probably through the spread of the pESI plasmid, treatment of this infection would be compromised.

Substrate Utilisation and Energy Metabolism in Non-Growing Campylobacter jejuni M1cam.

Campylobacter jejuni, the major cause of bacterial foodborne illness, is also a fastidious organism that requires strict growth requirements in the laboratory. Our aim was to study substrate utilisation and energy metabolism in non-growing C. jejuni to investigate the ability of these bacteria to survive so effectively in the food chain. We integrated phenotypic microarrays and genome-scale metabolic modelling (GSM) to investigate the survival of C. jejuni on 95 substrates. We further investigated the underlying metabolic re-adjustment associated with varying energy demands on each substrate. We identified amino acids, organic acids and H2, as single substrates supporting survival without growth. We identified several different mechanisms, which were used alone or in combination, for ATP production: substrate-level phosphorylation via acetate kinase, the TCA cycle, and oxidative phosphorylation via the electron transport chain that utilised alternative electron donors and acceptors. The benefit of ATP production through each of these mechanisms was associated with the cost of enzyme investment, nutrient availability and/or O2 utilisation. C. jejuni can utilise a wide range of substrates as energy sources, including organic acids commonly used for marination or preservation of ingredients, which might contribute to the success of their survival in changing environments.

Microbiomes of Urine and the Prostate Are Linked to Human Prostate Cancer Risk Groups.

BACKGROUND: Bacteria play a suspected role in the development of several cancer types, and associations between the presence of particular bacteria and prostate cancer have been reported. OBJECTIVE: To provide improved characterisation of the prostate and urine microbiome and to investigate the prognostic potential of the bacteria present. DESIGN, SETTING, AND PARTICIPANTS: Microbiome profiles were interrogated in sample collections of patient urine (sediment microscopy: n = 318, 16S ribosomal amplicon sequencing: n = 46; and extracellular vesicle RNA-seq: n = 40) and cancer tissue (n = 204). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Microbiomes were assessed using anaerobic culture, population-level 16S analysis, RNA-seq, and whole genome DNA sequencing. RESULTS AND LIMITATIONS: We demonstrate an association between the presence of bacteria in urine sediments and higher D'Amico risk prostate cancer (discovery, n = 215 patients, p < 0.001; validation, n = 103, p < 0.001, χ2 test for trend). Characterisation of the bacterial community led to the (1) identification of four novel bacteria (Porphyromonas sp. nov., Varibaculum sp. nov., Peptoniphilus sp. nov., and Fenollaria sp. nov.) that were frequently found in patient urine, and (2) definition of a patient subgroup associated with metastasis development (p = 0.015, log-rank test). The presence of five specific anaerobic genera, which includes three of the novel isolates, was associated with cancer risk group, in urine sediment (p = 0.045, log-rank test), urine extracellular vesicles (p = 0.039), and cancer tissue (p = 0.035), with a meta-analysis hazard ratio for disease progression of 2.60 (95% confidence interval: 1.39-4.85; p = 0.003; Cox regression). A limitation is that functional links to cancer development are not yet established. CONCLUSIONS: This study characterises prostate and urine microbiomes, and indicates that specific anaerobic bacteria genera have prognostic potential. PATIENT SUMMARY: In this study, we investigated the presence of bacteria in patient urine and the prostate. We identified four novel bacteria and suggest a potential prognostic utility for the microbiome in prostate cancer.

Genome-Scale Metabolic Modelling Approach to Understand the Metabolism of the Opportunistic Human Pathogen Staphylococcus epidermidis RP62A.

Staphylococcus epidermidis is a common commensal of collagen-rich regions of the body, such as the skin, but also represents a threat to patients with medical implants (joints and heart), and to preterm babies. Far less studied than Staphylococcus aureus, the mechanisms behind this increasingly recognised pathogenicity are yet to be fully understood. Improving our knowledge of the metabolic processes that allow S. epidermidis to colonise different body sites is key to defining its pathogenic potential. Thus, we have constructed a fully curated, genome-scale metabolic model for S. epidermidis RP62A, and investigated its metabolic properties with a focus on substrate auxotrophies and its utilisation for energy and biomass production. Our results show that, although glucose is available in the medium, only a small portion of it enters the glycolytic pathways, whils most is utilised for the production of biofilm, storage and the structural components of biomass. Amino acids, proline, valine, alanine, glutamate and arginine, are preferred sources of energy and biomass production. In contrast to previous studies, we have shown that this strain has no real substrate auxotrophies, although removal of proline from the media has the highest impact on the model and the experimental growth characteristics. Further study is needed to determine the significance of proline, an abundant amino acid in collagen, in S. epidermidis colonisation.

Quantitative bacterial counts in the bone marrow of Vietnamese patients with typhoid fever.

BACKGROUND: Bone marrow culture (BMC) is the reference standard for typhoid fever diagnosis. We studied the additional yield of BMC over blood culture (BC) and the relationship between quantitative BMC counts and severe disease. METHODS: Hospitalised Vietnamese patients with suspected typhoid fever were prospectively investigated with a BC, BMC, faecal culture and quantitative BMC counts. RESULTS: Salmonella typhi was isolated in 195 of 231 patients: from BC and BMC in 144 (73.8%), from BMC alone in 33 (16.9%), from BC alone in 12 (6.2%) and from faeces alone in 6 (3.1%). In 167 patients the median extracellular count of S. typhi was 2.5 cfu/mL (interquartile range [IQR] 0-10) and the intracellular count was 10.5 cfu/mL (IQR 2-42) with a ratio of 1.3 bacteria/cell (IQR 0.6-2.5). The median count of intracellular bacteria in 24 patients with severe disease was 46 bacteria/cell (IQR 9-105) compared with 6.5 bacteria/cell (IQR 2-34) in 143 with non-severe disease (p=0.005). The intracellular BMC count was negatively correlated with the peripheral white cell count and positively correlated with hepatomegaly, splenomegaly, aspartate transaminase, a positive BC and the fever clearance time following treatment with azithromycin, ofloxacin or a combination of the two. CONCLUSIONS: BMC gave a moderate additional yield over BC. Intracellular BMC counts may reflect the bacterial load in typhoid fever.

Massively parallel transposon mutagenesis identifies temporally essential genes for biofilm formation in Escherichia coli.

Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to colonize new environments. Progression through this life cycle requires temporally controlled gene expression to maximize fitness at each stage. Previous studies have largely focused on identifying genes essential for the formation of a mature biofilm; here, we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress, a massively parallel transposon mutagenesis approach using transposon-located promoters to assay the impact of disruption or altered expression of all genes in the genome on biofilm formation. We identified 48 genes that affected the fitness of cells growing in a biofilm, including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points, adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes, including some where expression changed between being beneficial or detrimental depending on the stage at which they are expressed, including dksA and dsbA. Novel genes implicated in biofilm formation included zapE and truA involved in cell division, maoP in chromosome organization, and yigZ and ykgJ of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.

Diagnostic Accuracy of Endobronchial Optical Coherence Tomography for the Microscopic Diagnosis of Usual Interstitial Pneumonia.

Rationale: Early, accurate diagnosis of interstitial lung disease (ILD) informs prognosis and therapy, especially in idiopathic pulmonary fibrosis (IPF). Current diagnostic methods are imperfect. High-resolution computed tomography has limited resolution, and surgical lung biopsy (SLB) carries risks of morbidity and mortality. Endobronchial optical coherence tomography (EB-OCT) is a low-risk, bronchoscope-compatible modality that images large lung volumes in vivo with microscopic resolution, including subpleural lung, and has the potential to improve the diagnostic accuracy of bronchoscopy for ILD diagnosis. Objectives: We performed a prospective diagnostic accuracy study of EB-OCT in patients with ILD with a low-confidence diagnosis undergoing SLB. The primary endpoints were EB-OCT sensitivity/specificity for diagnosis of the histopathologic pattern of usual interstitial pneumonia (UIP) and clinical IPF. The secondary endpoint was agreement between EB-OCT and SLB for diagnosis of the ILD fibrosis pattern. Methods: EB-OCT was performed immediately before SLB. The resulting EB-OCT images and histopathology were interpreted by blinded, independent pathologists. Clinical diagnosis was obtained from the treating pulmonologists after SLB, blinded to EB-OCT. Measurements and Main Results: We enrolled 31 patients, and 4 were excluded because of inconclusive histopathology or lack of EB-OCT data. Twenty-seven patients were included in the analysis (16 men, average age: 65.0 yr): 12 were diagnosed with UIP and 15 with non-UIP ILD. Average FVC and DlCO were 75.3% (SD, 18.5) and 53.5% (SD, 16.4), respectively. Sensitivity and specificity of EB-OCT was 100% (95% confidence interval, 75.8-100.0%) and 100% (79.6-100%), respectively, for both histopathologic UIP and clinical diagnosis of IPF. There was high agreement between EB-OCT and histopathology for diagnosis of ILD fibrosis pattern (weighted κ: 0.87 [0.72-1.0]). Conclusions: EB-OCT is a safe, accurate method for microscopic ILD diagnosis, as a complement to high-resolution computed tomography and an alternative to SLB.

Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management.

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.