Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.

We tested a technique (FT-IR) to identify four different, common bacteria from 104 children with serious infections and compared it to lab methods for diagnosis. FT-IR was more accurate. We tested if it could identify subtypes of bacteria, which is important in outbreaks. It was able to subtype two species, but not the two other species. However, it is a much faster and cheaper technique than the gold standard. It may be useful in certain outbreaks. We also investigated the trends between genes and the length of hospital stay. This can support further laboratory research. As a fast, low-cost test, FT-IR warrants further testing before it is applied to clinical labs.

Corrigendum: Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

[This corrects the article DOI: 10.3389/fmicb.2024.1334268.].

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

Introduction: The emergence of multi-drug resistant (MDR) pathogens linked to healthcare-associated infections (HCAIs) is an increasing concern in modern veterinary practice. Thus, rapid bacterial typing for real-time tracking of MDR hospital dissemination is still much needed to inform best infection control practices in a clinically relevant timeframe. To this end, the IR Biotyper using Fourier-Transform InfraRed (FTIR) spectroscopy has the potential to provide fast cluster analysis of potentially related organisms with substantial cost and turnaround time benefits. Materials and methods: A collection of MDR bacterial isolates (n = 199, comprising 92 Klebsiella pneumoniae and 107 Pseudomonas aeruginosa) obtained from companion animal (i.e., dogs, cats and horses) clinical investigations, faecal and environmental screening from four veterinary facilities between 2012 and 2019 was analysed retrospectively by FTIR spectroscopy. Its performance was compared against MLST extracted from whole genomes of a subset of clustering isolates (proportionally to cluster size) for investigation of potential nosocomial transmission between patients and the surrounding hospital environments. Results: Concordance between the FTIR and MLST types was overall high for K. pneumoniae (Adjusted Rand Index [ARI] of 0.958) and poor for P. aeruginosa (ARI of 0.313). FTIR K. pneumoniae clusters (n = 7) accurately segregated into their respective veterinary facility with evidence of intra-hospital spread of K. pneumoniae between patients and environmental surfaces. Notably, K. pneumoniae ST147 intensely circulated at one Small Animal Hospital ICU. Conversely, Pseudomonas aeruginosa FTIR clusters (n = 18) commonly contained isolates of diversified hospital source and heterogeneous genetic background (as also genetically related isolates spread across different clusters); nonetheless, dissemination of some clones, such as P. aeruginosa ST2644 in the equine hospital, was apparent. Importantly, FTIR clustering of clinical, colonisation and/or environmental isolates sharing genomically similar backgrounds was seen for both MDR organisms, highlighting likely cross-contamination events that led to clonal dissemination within settings. Conclusion: FTIR spectroscopy has high discriminatory power for hospital epidemiological surveillance of veterinary K. pneumoniae and could provide sufficient information to support early detection of clonal dissemination, facilitating implementation of appropriate infection control measures. Further work and careful optimisation need to be carried out to improve its performance for typing of P. aeruginosa veterinary isolates.

Genomic profiling of idiopathic peri-hilar cholangiocarcinoma reveals new targets and mutational pathways.

Peri-hilar cholangiocarcinoma (pCCA) is chemorefractory and limited genomic analyses have been undertaken in Western idiopathic disease. We undertook comprehensive genomic analyses of a U.K. idiopathic pCCA cohort to characterize its mutational profile and identify new targets. Whole exome and targeted DNA sequencing was performed on forty-two resected pCCA tumors and normal bile ducts, with Gene Set Enrichment Analysis (GSEA) using one-tailed testing to generate false discovery rates (FDR). 60% of patients harbored one cancer-associated mutation, with two mutations in 20%. High frequency somatic mutations in genes not typically associated with cholangiocarcinoma included mTOR, ABL1 and NOTCH1. We identified non-synonymous mutation (p.Glu38del) in MAP3K9 in ten tumors, associated with increased peri-vascular invasion (Fisher's exact, p < 0.018). Mutation-enriched pathways were primarily immunological, including innate Dectin-2 (FDR 0.001) and adaptive T-cell receptor pathways including PD-1 (FDR 0.007), CD4 phosphorylation (FDR 0.009) and ZAP70 translocation (FDR 0.009), with overlapping HLA genes. We observed cancer-associated mutations in over half of our patients. Many of these mutations are not typically associated with cholangiocarcinoma yet may increase eligibility for contemporary targeted trials. We also identified a targetable MAP3K9 mutation, in addition to oncogenic and immunological pathways hitherto not described in any cholangiocarcinoma subtype.

Panel sequencing links rare, likely damaging gene variants with distinct clinical phenotypes and outcomes in juvenile-onset SLE.

OBJECTIVES: Juvenile-onset systemic lupus erythematosus (jSLE) affects 15-20% of lupus patients. Clinical heterogeneity between racial groups, age groups and individual patients suggests variable pathophysiology. This study aimed to identify highly penetrant damaging mutations in genes associated with SLE/SLE-like disease in a large national cohort (UK JSLE Cohort Study) and compare demographic, clinical and laboratory features in patient sub-cohorts with 'genetic' SLE vs remaining SLE patients. METHODS: Based on a sequencing panel designed in 2018, target enrichment and next-generation sequencing were performed in 348 patients to identify damaging gene variants. Findings were integrated with demographic, clinical and treatment related datasets. RESULTS: Damaging gene variants were identified in ∼3.5% of jSLE patients. When compared with the remaining cohort, 'genetic' SLE affected younger children and more Black African/Caribbean patients. 'Genetic' SLE patients exhibited less organ involvement and damage, and neuropsychiatric involvement developed over time. Less aggressive first line treatment was chosen in 'genetic' SLE patients, but more second and third line agents were used. 'Genetic' SLE associated with anti-dsDNA antibody positivity at diagnosis and reduced ANA, anti-LA and anti-Sm antibody positivity at last visit. CONCLUSION: Approximately 3.5% of jSLE patients present damaging gene variants associated with younger age at onset, and distinct clinical features. As less commonly observed after treatment induction, in 'genetic' SLE, autoantibody positivity may be the result of tissue damage and explain reduced immune complex-mediated renal and haematological involvement. Routine sequencing could allow for patient stratification, risk assessment and target-directed treatment, thereby increasing efficacy and reducing toxicity.

Effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control for hospital-onset COVID-19 infection: Multicentre, prospective study.

Background: Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods: We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48 hr) and 4 weeks of 'longer-turnaround' (5-10 days) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital-onset COVID-19 infections (HOCIs; detected ≥48 hr from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on the incidence of probable/definite hospital-acquired infections (HAIs), was evaluated. Results: A total of 2170 HOCI cases were recorded from October 2020 to April 2021, corresponding to a period of extreme strain on the health service, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (incidence rate ratio 1.60, 95% CI 0.85-3.01; p=0.14) or rapid (0.85, 0.48-1.50; p=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8 and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2 and 11.6% of cases where the report was returned. In a 'per-protocol' sensitivity analysis, there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Capacity to respond effectively to insights from sequencing was breached in most sites by the volume of cases and limited resources. Conclusions: While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days. Funding: COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (grant code: MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. Clinical trial number: NCT04405934.

Generation and transmission of interlineage recombinants in the SARS-CoV-2 pandemic.

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single-nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances, there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of 2 months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7's set of mutations.

Campylobacter Contamination at Retail of Halal Chicken Produced in the United Kingdom.

ABSTRACT: Campylobacter is the leading cause of human bacterial diarrheal disease worldwide, and poultry meat products account for the majority of human cases. Based on recent surveys, the Food Standards Agency has estimated the Campylobacter prevalence in fresh retail chicken in the United Kingdom to be 41.2%. However, such surveys have not distinguished between broiler chickens produced for different consumer demographic groups, such as the Halal market. Campylobacter colonization of broilers is difficult to prevent, especially during routine partial depopulation of flocks. Broilers produced for the Halal market may undergo multiple depopulation events, which may increase the risk of Campylobacter colonization and subsequent contamination of chicken meat. This study was conducted to determine the prevalence and levels of Campylobacter contamination in chicken meat produced for the Halal market in the United Kingdom. Campylobacter was identified and enumerated from the neck skin and outer packaging of 405 Halal chickens. Culture isolates were assigned to species via PCR assays, and disk diffusion assays were used to determine antimicrobial susceptibility. Logistic regression analysis was used to assess risk factors for Campylobacter contamination, the level of Campylobacter contamination among positive carcasses, and antimicrobial resistance. Campylobacter spp. were confirmed in 65.4% of neck skin samples and 17.1% of packaging samples. Neck skin samples had the highest level of contamination; 13.8% of samples had >1,000 CFU/g. Large birds had a significantly higher number of samples with >1,000 CFU/g (P < 0.001). and as chicken carcass weight increased, birds were more likely to be Campylobacter positive (P < 0.05). A high prevalence of resistance was seen to ciprofloxacin (42.0% of samples), and 38.5% of samples contained at least one multidrug-resistant Campylobacter isolate. This study revealed that Halal chicken has a higher Campylobacter prevalence than does non-Halal chicken. Interventions should be introduced to reduce this public health risk.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Outbreak of Severe Vomiting in Dogs Associated with a Canine Enteric Coronavirus, United Kingdom.

The lack of population health surveillance for companion animal populations leaves them vulnerable to the effects of novel diseases without means of early detection. We present evidence on the effectiveness of a system that enabled early detection and rapid response a canine gastroenteritis outbreak in the United Kingdom. In January 2020, prolific vomiting among dogs was sporadically reported in the United Kingdom. Electronic health records from a nationwide sentinel network of veterinary practices confirmed a significant increase in dogs with signs of gastroenteric disease. Male dogs and dogs living with other vomiting dogs were more likely to be affected. Diet and vaccination status were not associated with the disease; however, a canine enteric coronavirus was significantly associated with illness. The system we describe potentially fills a gap in surveillance in neglected populations and could provide a blueprint for other countries.

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

Targeted Next-Generation Sequencing of 117 Routine Clinical Samples Provides Further Insights into the Molecular Landscape of Uveal Melanoma.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX, most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX, as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1, and SF3B1 and EIF1AX, respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.

Increasing prevalence of a fluoroquinolone resistance mutation amongst Campylobacter jejuni isolates from four human infectious intestinal disease studies in the United Kingdom.

BACKGROUND: Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease. METHODS: We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993-1996; n = 293] and IID2 [isolates from 2008-2009; n = 93]), the INTEGRATE project [isolates from 2016-2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163]. RESULTS: There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation. DISCUSSION: Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation.

Immunological observations and transcriptomic analysis of trimester-specific full-term placentas from three Zika virus-infected women.

OBJECTIVES: Effects of Zika virus (ZIKV) infection on placental development during pregnancy are unclear. METHODS: Full-term placentas from three women, each infected with ZIKV during specific pregnancy trimesters, were harvested for anatomic, immunologic and transcriptomic analysis. RESULTS: In this study, each woman exhibited a unique immune response with raised IL-1RA, IP-10, EGF and RANTES expression and neutrophil numbers during the acute infection phase. Although ZIKV NS3 antigens co-localised to placental Hofbauer cells, the placentas showed no anatomic defects. Transcriptomic analysis of samples from the placentas revealed that infection during trimester 1 caused a disparate cellular response centred on differential eIF2 signalling, mitochondrial dysfunction and oxidative phosphorylation. Despite these, the babies were delivered without any congenital anomalies. CONCLUSION: These findings should translate to improve clinical prenatal screening procedures for virus-infected pregnant patients.

Draft Genome Assembly of the Poultry Red Mite, Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae, is a major worldwide concern in the egg-laying industry. Here, we report the first draft genome assembly and gene prediction of Dermanyssus gallinae, based on combined PacBio and MinION long-read de novo sequencing. The ∼959-Mb genome is predicted to encode 14,608 protein-coding genes.

Draft Genome Assembly of the Sheep Scab Mite, Psoroptes ovis.

Sheep scab, caused by infestation with Psoroptes ovis, is highly contagious, results in intense pruritus, and represents a major welfare and economic concern. Here, we report the first draft genome assembly and gene prediction of P. ovis based on PacBio de novo sequencing. The ∼63.2-Mb genome encodes 12,041 protein-coding genes.

Genomics of habitat choice and adaptive evolution in a deep-sea fish.

Intraspecific diversity promotes evolutionary change, and when partitioned among geographic regions or habitats can form the basis for speciation. Marine species live in an environment that can provide as much scope for diversification in the vertical as in the horizontal dimension. Understanding the relevant mechanisms will contribute significantly to our understanding of eco-evolutionary processes and effective biodiversity conservation. Here, we provide an annotated genome assembly for the deep-sea fish Coryphaenoides rupestris and re-sequencing data to show that differentiation at non-synonymous sites in functional loci distinguishes individuals living at different depths, independent of horizontal spatial distance. Our data indicate disruptive selection at these loci; however, we find no clear evidence for differentiation at neutral loci that may indicate assortative mating. We propose that individuals with distinct genotypes at relevant loci segregate by depth as they mature (supported by survey data), which may be associated with ecotype differentiation linked to distinct phenotypic requirements at different depths.

Transmission and lineage displacement drive rapid population genomic flux in cystic fibrosis airway infections of a Pseudomonas aeruginosa epidemic strain.

Pseudomonas aeruginosa chronic infections of cystic fibrosis (CF) airways are a paradigm for within-host evolution with abundant evidence for rapid evolutionary adaptation and diversification. Recently emerged transmissible strains have spread globally, with the Liverpool Epidemic Strain (LES) the most common strain infecting the UK CF population. Previously we have shown that highly divergent lineages of LES can be found within a single infection, consistent with super-infection among a cross-sectional cohort of patients. However, despite its clinical importance, little is known about the impact of transmission on the genetic structure of these infections over time. To characterize this, we longitudinally sampled a meta-population of 15 genetic lineages within the LES over 13 months among seven chronically infected CF patients by genome sequencing. Comparative genome analyses of P. aeruginosa populations revealed that the presence of coexisting lineages contributed more to genetic diversity within an infection than diversification in situ. We observed rapid and substantial shifts in the relative abundance of lineages and replacement of dominant lineages, likely to represent super-infection by repeated transmissions. Lineage dynamics within patients led to rapid changes in the frequencies of mutations across suites of linked loci carried by each lineage. Many loci were associated with important infection phenotypes such as antibiotic resistance, mucoidy and quorum sensing, and were repeatedly mutated in different lineages. These findings suggest that transmission leads to rapid shifts in the genetic structure of CF infections, including in clinically important phenotypes such as antimicrobial resistance, and is likely to impede accurate diagnosis and treatment.

Identification of Equid herpesvirus 2 in tissue-engineered equine tendon.

Background: Incidental findings of virus-like particles were identified following electron microscopy of tissue-engineered tendon constructs (TETC) derived from equine tenocytes. We set out to determine the nature of these particles, as there are few studies which identify virus in tendons per se, and their presence could have implications for tissue-engineering using allogenic grafts. Methods: Virus particles were identified in electron microscopy of TETCs. Virion morphology was used to initially hypothesise the virus identity.  Next generation sequencing was implemented to identify the virus. A pan herpesvirus PCR was used to validate the RNASeq findings using an independent platform. Histological analysis and biochemical analysis was undertaken on the TETCs. Results: Morphological features suggested the virus to be either a retrovirus or herpesvirus. Subsequent next generation sequencing mapped reads to Equid herpesvirus 2 (EHV2). Histological examination and biochemical testing for collagen content revealed no significant differences between virally affected TETCs and non-affected TETCs. An independent set of equine superficial digital flexor tendon tissue (n=10) examined using designed primers for specific EHV2 contigs identified at sequencing were negative. These data suggest that EHV is resident in some equine tendon. Conclusions: EHV2 was demonstrated in equine tenocytes for the first time; likely from in vivo infection. The presence of EHV2 could have implications to both tissue-engineering and tendinopathy.