Regulation of a phage defence island by RptR, a novel repressor that controls restriction-modification systems in diverse bacteria

Bacteria encode a panoply of defence systems to overcome phage infection. In recent years, over 100 defence systems have been identified, with the majority of these found co-localized in defence islands. Although there has been much progress in understanding the mechanisms of anti-phage defence employed by bacteria, far less is known about their regulation before and during phage infection. Here, we describe RptR (RMS-proximal transcriptional regulator), a small transcriptional regulator of a defence island in enteropathogenic Escherichia coli composed of a toxin-antitoxin system, DarTG2, embedded within a Type I restriction-modification system (RMS). We determined the molecular structure of a RptR homodimer and, using transcriptional reporter and in vitro DNA binding assays, show that RptR represses the promoter of the defence island by binding to a series of three direct repeats in the promoter. Furthermore, we demonstrate, using the structural models of RptR validated with electrophoretic mobility shift assays, that the minimal RptR binding site is a 6-bp palindrome, TAGCTA. Both RptR and its binding site are highly conserved across diverse bacterial genomes with a strong genetic association with Type I RMS, highlighting the role of RptR as a novel regulatory component of an important mechanism for anti-phage defence in bacteria

Shared mechanisms of enhanced plasmid maintenance and antibiotic tolerance mediated by the VapBC toxin:antitoxin system

Toxin:antitoxin (TA) systems are widespread in bacteria and were first identified as plasmid addiction systems that kill bacteria lacking a TA-encoding plasmid following cell division. TA systems have also been implicated in bacterial persistence and antibiotic tolerance, which can be precursors of antibiotic resistance. Here, we identified a clinical isolate of Shigella sonnei (CS14) with a remarkably stable pINV virulence plasmid; pINV is usually frequently lost from S. sonnei, but plasmid loss was not detected from CS14. We found that the plasmid in CS14 is stabilized by a single nucleotide polymorphism (SNP) in its vapBC TA system. VapBC TA systems are the most common Type II TA system in bacteria, and consist of a VapB antitoxin and VapC PIN domain-containing toxin. The plasmid stabilizing SNP leads to a Q12L substitution in the DNA-binding domain of VapB, which reduces VapBC binding to its own promoter, impairing vapBC autorepression. However, VapBL12C mediates high-level plasmid stabilization because VapBL12 is more prone to degradation by Lon than wild-type VapB; this liberates VapC to efficiently kill bacteria that no longer contain a plasmid. Of note, mutations that confer tolerance to antibiotics in Escherichia coli also map to the DNA-binding domain of VapBC encoded by the chromosomally integrated F plasmid. We demonstrate that the tolerance mutations also enhance plasmid stabilization by the same mechanism as VapBL12. Our findings highlight the links between plasmid maintenance and antibiotic tolerance, both of which can promote the development of antimicrobial resistance

Bacteriocin-like peptides encoded by a horizontally acquired island mediate Neisseria gonorrhoeae autolysis

Neisseria gonorrhoeae is a human-specific pathogen that causes the important sexually transmitted infection, gonorrhoea, an inflammatory condition of the genitourinary tract. The bacterium is closely related to the meningococcus, a leading cause of bacterial meningitis. Both these invasive bacterial species undergo autolysis when in the stationary phase of growth. Autolysis is a form of programmed cell death (PCD) which is part of the life cycle of remarkably few bacteria and poses an evolutionary conundrum as altruistic death provides no obvious benefit for single-celled organisms. Here, we searched for genes present in these 2 invasive species but not in other members of the Neisseria genus. We identified a ~3.4 kb horizontally acquired region, we termed the nap island, which is largely restricted to the gonococcus and meningococcus. The nap island in the gonococcus encodes 3 cationic, bacteriocin-like peptides which have no detectable antimicrobial activity. Instead, the gonococcal Neisseria autolysis peptides (Naps) promote autolytic cell death when bacteria enter the stationary phase of growth. Furthermore, strains lacking the Naps exhibit reduced autolysis in assays of PCD. Expression of Naps is likely to be phase variable, explaining how PCD could have arisen in these important human pathogens. NapC also induces lysis of human cells, so the peptides are likely to have multiple roles during colonisation and disease. The acquisition of the nap island contributed to the emergence of PCD in the gonococcus and meningococcus and potentially to the appearance of invasive disease in Neisseria spp

Functional Significance of Factor H Binding to <i>Neisseria meningitidis</i>

Abstract Neisseria meningitidis is an important cause of septicemia and meningitis. To cause disease, the bacterium must successfully survive in the bloodstream where it has to avoid being killed by host innate immune mechanisms, particularly the complement system. A number of pathogenic microbes bind factor H (fH), the negative regulator of the alternative pathway of complement activation, to promote their survival in vivo. In this study, we show that N. meningitidis binds fH to its surface. Binding to serogroups A, B, and C N. meningitidis strains was detected by FACS and Far Western blot analysis, and occurred in the absence of other serum factors such as C3b. Unlike Neisseria gonorrhoeae, binding of fH to N. meningitidis was independent of sialic acid on the bacterium, either as a component of its LPS or its capsule. Characterization of the major fH binding partner demonstrated that it is a 33-kDa protein; examination of insertion mutants showed that porins A and B, outer membrane porins expressed by N. meningitidis, do not contribute significantly to fH binding. We examined the physiological consequences of fH bound to the bacterial surface. We found that fH retains its activity as a cofactor of factor I when bound to the bacterium and contributes to the ability of N. meningitidis to avoid complement-mediated killing in the presence of human serum. Therefore, the recruitment of fH provides another mechanism by which this important human pathogen evades host innate immunity.</jats:p

Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules.

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps

Towards a standardized protocol to assess natural capital and ecosystem services in solar parks

1. Natural capital and ecosystem services have emerged as fundamental concepts of ecosystem management strategies in the past two decades, particularly within major international land assessment frameworks, including the UN's Millennium Ecosystem Assessment and the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services' Global Assessment Report. 2. Despite the recent development of several analytical methods and models to quantify changes in natural capital and ecosystem services resulting from land use change, incorporating them into the land planning process can be challenging from a practical point of view without guidance on standard methods. 3. In an attempt to decarbonize energy supply systems to meet internationally agreed targets on climate change, solar energy production, in the form of ground-mounted solar parks, is emerging as one of the dominant forms of temporary land use for renewable energies globally. 4. We propose 19 directly measurable indicators associated with 16 ecosystem services within three major stocks of natural capital (biodiversity, soil and water) that are most likely to be impacted by the development of solar parks. Indicators are supported by well-established methods that have been widely used in pure and applied land use research within terrestrial ecosystems. Moreover, they can be implemented flexibly according to interest or land management objectives. 5. Whilst not intended as a precise recipe for how to assess the effects of solar park development on hosting ecosystems, the protocol will guide the solar energy industry and all actors involved, be they researchers, practitioners, ecological consultancies or statutory bodies, to implement a standardized approach to evaluate temporal and spatial changes in natural capital and ecosystem services resulting from solar park development and operation, with the ultimate aim of generating comparable and reproducible data on ecosystem impact assessment across the solar energy sector

Environmental factors associated with general practitioner consultations for allergic rhinitis in London, England: a retrospective time series analysis

Objectives: To identify key predictors of general practitioner (GP) consultations for allergic rhinitis (AR) using meteorological and environmental data. Design: A retrospective, time series analysis of GP consultations for AR. Setting: A large GP surveillance network of GP practices in the London area. Participants: The study population was all persons who presented to general practices in London that report to the Public Health England GP in-hours syndromic surveillance system during the study period (3 April 2012 to 11 August 2014). Primary measure: Consultations for AR (numbers of consultations). Results: During the study period there were 186 401 GP consultations for AR. High grass and nettle pollen counts (combined) were associated with the highest increases in consultations (for the category 216-270 grains/m3, relative risk (RR) 3.33, 95% CI 2.69 to 4.12) followed by high tree (oak, birch and plane combined) pollen counts (for the category 260–325 grains/m3, RR 1.69, 95% CI 1.32 to 2.15) and average daily temperatures between 15°C and 20°C (RR 1.47, 95% CI 1.20 to 1.81). Higher levels of nitrogen dioxide (NO2) appeared to be associated with increased consultations (for the category 70–85 µg/m3, RR 1.33, 95% CI 1.03 to 1.71), but a significant effect was not found with ozone. Higher daily rainfall was associated with fewer consultations (15–20 mm/day; RR 0.812, 95% CI 0.674 to 0.980). Conclusions: Changes in grass, nettle or tree pollen counts, temperatures between 15°C and 20°C, and (to a lesser extent) NO2 concentrations were found to be associated with increased consultations for AR. Rainfall has a negative effect. In the context of climate change and continued exposures to environmental air pollution, intelligent use of these data will aid targeting public health messages and plan healthcare demand

OrthoticS for TReatment of symptomatic flat feet In CHildren (OSTRICH):a randomised controlled trial

Background Children and young people with symptomatic pes planus (flat feet) often seek treatment from healthcare professionals. There are various treatment options, but there is a lack of high-quality evidence about which is most effective. Objectives To assess the clinical and cost-effectiveness of prefabricated orthoses, plus exercise and advice, compared with exercise and advice alone on physical function, measured using the physical domain of the Oxford Ankle Foot Questionnaire for Children, among children with symptomatic pes planus. Design and methods A pragmatic, multicentre, two-armed individually randomised controlled trial with an internal pilot, economic evaluation and qualitative study. Setting and participants Children and young people aged 6–14 years with symptomatic flat feet were recruited from hospital or community healthcare facilities in England and Wales. Participants were randomised 1 : 1 using a secure web-based randomisation system and followed up for up to 12 months. Interventions We planned to provide all participants with advice and exercises, with the intervention group also receiving a prefabricated orthosis. Due to the nature of the study treatments, blinding of participants or the research team was not possible. Main outcome measures The primary outcome was the physical domain subscale of the Oxford Ankle Foot Questionnaire for Children over the 12-month follow-up. Secondary outcomes included the physical domain subscale at 3, 6 and 12 months, and the ‘School and Play’ and ‘Emotional’ domains of the Oxford Ankle Foot Questionnaire, pain scores, healthcare resource use, EQ-5D-Y and Child Health Utility 9D at all time points. The qualitative study drew on health literacy and health belief perspectives and examined fidelity and explored the experiences of being in the trial for those receiving and delivering the study treatments. Results COVID-19 severely delayed trial set-up and recruitment and the study closed before meeting its recruitment target. Of 549 participants assessed for eligibility, 134 were randomised (intervention n = 70, control n = 64). The mean age of participants was 10.6 years (range 6.3–14.8) and 55.2% were male. No adverse events were reported. The planned statistical and health economic analyses could not be fully conducted due to the limited data. The qualitative study identified pain, posture and gait as the most common concerns by participants with pain relief as the primary motivator for seeking health care. Participants generally reported little understanding of their condition with barriers including misattribution (e.g. growing pains). Misinformation was common emphasising a need for accessible accurate education materials and structured follow-up care. There was a common belief that orthoses were superior to exercises leading to high levels of adherence, satisfaction and outcomes with orthoses compared with poor adherence, and low perceived efficacy with exercises linked to challenges incorporating these into daily routines. Limitations We could not deliver the study objectives as planned. Due to the limited data available, we were unable to undertake the planned analysis. Conclusions The COVID-19 pandemic significantly impacted trial set-up and recruitment. Extending the study was not feasible due to cost and time constraints. Future work The evidence for the clinical and cost-effectiveness of orthotics for the treatment of symptomatic flat feet in children remains inconclusive and an area for further research. Funding This synopsis presents independent research funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme as award number NIHR127510

OrthoticS for TReatment of symptomatic flat feet In CHildren (OSTRICH): a randomised controlled trial

BackgroundChildren and young people with symptomatic pes planus (flat feet) often seek treatment from healthcare professionals. There are various treatment options, but there is a lack of high-quality evidence about which is most effective.ObjectivesTo assess the clinical and cost-effectiveness of prefabricated orthoses, plus exercise and advice, compared with exercise and advice alone on physical function, measured using the physical domain of the Oxford Ankle Foot Questionnaire for Children, among children with symptomatic pes planus.Design and methodsA pragmatic, multicentre, two-armed individually randomised controlled trial with an internal pilot, economic evaluation and qualitative study.Setting and participantsChildren and young people aged 6–14 years with symptomatic flat feet were recruited from hospital or community healthcare facilities in England and Wales. Participants were randomised 1 : 1 using a secure web-based randomisation system and followed up for up to 12 months.InterventionsWe planned to provide all participants with advice and exercises, with the intervention group also receiving a prefabricated orthosis. Due to the nature of the study treatments, blinding of participants or the research team was not possible.Main outcome measuresThe primary outcome was the physical domain subscale of the Oxford Ankle Foot Questionnaire for Children over the 12-month follow-up. Secondary outcomes included the physical domain subscale at 3, 6 and 12 months, and the ‘School and Play’ and ‘Emotional’ domains of the Oxford Ankle Foot Questionnaire, pain scores, healthcare resource use, EQ-5D-Y and Child Health Utility 9D at all time points. The qualitative study drew on health literacy and health belief perspectives and examined fidelity and explored the experiences of being in the trial for those receiving and delivering the study treatments.ResultsCOVID-19 severely delayed trial set-up and recruitment and the study closed before meeting its recruitment target. Of 549 participants assessed for eligibility, 134 were randomised (intervention n = 70, control n = 64). The mean age of participants was 10.6 years (range 6.3–14.8) and 55.2% were male. No adverse events were reported. The planned statistical and health economic analyses could not be fully conducted due to the limited data. The qualitative study identified pain, posture and gait as the most common concerns by participants with pain relief as the primary motivator for seeking health care. Participants generally reported little understanding of their condition with barriers including misattribution (e.g. growing pains). Misinformation was common emphasising a need for accessible accurate education materials and structured follow-up care. There was a common belief that orthoses were superior to exercises leading to high levels of adherence, satisfaction and outcomes with orthoses compared with poor adherence, and low perceived efficacy with exercises linked to challenges incorporating these into daily routines.LimitationsWe could not deliver the study objectives as planned. Due to the limited data available, we were unable to undertake the planned analysis.ConclusionsThe COVID-19 pandemic significantly impacted trial set-up and recruitment. Extending the study was not feasible due to cost and time constraints.Future workThe evidence for the clinical and cost-effectiveness of orthotics for the treatment of symptomatic flat feet in children remains inconclusive and an area for further research

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research