The tier 1 bioterrorism agent Burkholderia pseudomallei causes melioidosis, a tropical disease with fatality rates that can exceed 40% despite antibiotic therapy. Antibiotic failure is likely to be, at least in part, due to biofilm-dwelling B. pseudomallei, and therefore, an improved understanding of how this pathogen regulates biofilm formation could reveal new opportunities for clinical intervention. The antimicrobial radical nitric oxide (NO) plays a key role in host immune defenses against bacteria, and the ability of B. pseudomallei to sense and mitigate NO toxicity is vital for establishing infection. NO-sensing proteins (NosPs), which have recently emerged as key regulators of biofilm formation in many bacterial species, use a FIST domain to sense NO via a bound heme. We hypothesized that the NosP homolog in B. pseudomallei would regulate biofilm formation and mediate NO-protective responses. We used [γ-32P]ATP autophosphorylation assays to show that NosP of B. pseudomallei controls the autophosphorylation rate of an associated histidine kinase protein (NosK) in an NO-dependent manner. NosK was found to phosphorylate a response regulator protein (NosR) with an HD-GYP output domain, which is associated with c-di-GMP signaling, therefore implicating NosP in modulating c-di-GMP-regulated phenotypes. Unmarked, in-frame deletion of either nosP or nosK caused significant changes in B. pseudomallei biofilm formation and increased sensitivity to nitrosative stress, in addition to affecting other virulence traits such as growth and swimming motility. These results indicate that NosP and NosK signaling control a range of infection-relevant phenotypes and may serve as targets for novel therapeutic intervention.IMPORTANCEMelioidosis is an emerging, potentially life-threatening infection caused by the bacterium Burkholderia pseudomallei, killing ~89,000 people per year globally. Antibiotic therapy fails in ~10%-40% of cases, and hence, an improved understanding of the molecular mechanisms that control B. pseudomallei virulence could reveal new approaches for improving melioidosis treatment. Biofilm formation and resistance to the antimicrobial radical NO are virulence traits that help bacteria establish infections. Here, we show that two proteins in B. pseudomallei, NosP and NosK, work together to detect NO and regulate a suite of virulence traits, including NO resistance, biofilm formation, growth, and swimming motility. This work, therefore, improves our understanding of the molecular mechanisms that control infection-related phenotypes in B. pseudomallei.

Antimicrobial resistance poses one of the greatest threats to global health and there is an urgent need for new therapeutic options. Phages are viruses that infect and kill bacteria and phage therapy could provide a valuable tool for the treatment of multidrug-resistant infections. In this study, water samples collected by citizen scientists as part of the Citizen Phage Library (CPL) project, and wastewater samples from the Environment Agency yielded phages with activity against clinical strains Klebsiella pneumoniae BPRG1484 and Enterobacter cloacae BPRG1482. A total of 169 and 163 phages were found for K. pneumoniae and E. cloacae, respectively, within four days of receiving the strains. A third strain (Escherichia coli BPRG1486) demonstrated cross-reactivity with 42 E. coli phages already held in the CPL collection. Seed lots were prepared for four K. pneumoniae phages and a cocktail combining these phages was found to reduce melanisation in a Galleria mellonella infection model. The resources and protocols utilised by the Citizen Phage Library enabled the rapid isolation and characterisation of phages targeted against multiple strains. In the future, within a clearly defined regulatory framework, phage therapy could be made available on a named-patient basis within the UK.

Introduction. Diabetic foot infection (DFI) is the main reason for diabetes- related hospitalisation and is a major cause of diabetes- related amputation. DFIs are often complicated by ischaemia in the affected limb, the presence of polymicrobial biofilms and increasingly the occurrence of antibiotic resistant bacteria. Hypothesis/Gap statement. Antibiotic loaded beads could inhibit the growth of polymicrobial DFI communities with differing compositions in vitro. Aim. This study investigates the in vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure, Biocomposites Ltd., UK) against polymicrobial DFI communities and individual bacterial strains derived from DFIs. Methodology. Debrided tissue obtained from the base of infected diabetic foot ulcers was homogenised and spread over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates. Calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar and following incubation, zones of inhibition (ZOI) were measured. For individual bacterial strains isolated from the infected tissue, calcium sulfate beads containing vancomycin, gentamicin, flucloxacillin or rifampicin and beads containing a combination of vancomycin and gentamicin or flucloxacillin and rifampicin were tested for their ability to inhibit growth. Results. Calcium sulfate beads loaded with a combination of vancomycin and gentamicin were able to inhibit bacterial growth from all polymicrobial tissue homogenates tested, with ZOI diameters ranging from 15 to 40 mm. In the case of individual bacterial strains, beads containing combinations of vancomycin and gentamicin or flucloxacillin and rifampicin were able to produce ZOI with Gram- positive facultatitive anaerobic strains such as Staphylococcus aureus and Enterococcus faecalis, Gram-negative facultative anaerobic strains such as Pseudomonas aeruginosa and obligate anaerobic strains such as Finegoldia magna even where acquired resistance to one of the antibiotics in the combination was evidenced. Conclusion. The local use of calcium sulfate beads containing a combination of two antibiotics demonstrated high efficacy against polymicrobial DFI communities and individual DFI bacterial strains in in vitro zone of inhibition tests. These results show promise for clinical application, but further research and clinical studies are required.

OBJECTIVE:Antitumour necrosis factor (TNF) drugs impair serological responses following SARS-CoV-2 vaccination. We sought to assess if a third dose of a messenger RNA (mRNA)-based vaccine substantially boosted anti-SARS-CoV-2 antibody responses and protective immunity in infliximab-treated patients with IBD. DESIGN:Third dose vaccine induced anti-SARS-CoV-2 spike (anti-S) receptor-binding domain (RBD) antibody responses, breakthrough SARS-CoV-2 infection, reinfection and persistent oropharyngeal carriage in patients with IBD treated with infliximab were compared with a reference cohort treated with vedolizumab from the impaCt of bioLogic therApy on saRs-cov-2 Infection and immuniTY (CLARITY) IBD study. RESULTS:Geometric mean (SD) anti-S RBD antibody concentrations increased in both groups following a third dose of an mRNA-based vaccine. However, concentrations were lower in patients treated with infliximab than vedolizumab, irrespective of whether their first two primary vaccine doses were ChAdOx1 nCoV-19 (1856 U/mL (5.2) vs 10 728 U/mL (3.1), p<0.0001) or BNT162b2 vaccines (2164 U/mL (4.1) vs 15 116 U/mL (3.4), p<0.0001). However, no differences in anti-S RBD antibody concentrations were seen following third and fourth doses of an mRNA-based vaccine, irrespective of the combination of primary vaccinations received. Post-third dose, anti-S RBD antibody half-life estimates were shorter in infliximab-treated than vedolizumab-treated patients (37.0 days (95% CI 35.6 to 38.6) vs 52.0 days (95% CI 49.0 to 55.4), p<0.0001).Compared with vedolizumab-treated, infliximab-treated patients were more likely to experience SARS-CoV-2 breakthrough infection (HR 2.23 (95% CI 1.46 to 3.38), p=0.00018) and reinfection (HR 2.10 (95% CI 1.31 to 3.35), p=0.0019), but this effect was uncoupled from third vaccine dose anti-S RBD antibody concentrations. Reinfection occurred predominantly during the Omicron wave and was predicted by SARS-CoV-2 antinucleocapsid concentrations after the initial infection. We did not observe persistent oropharyngeal carriage of SARS-CoV-2. Hospitalisations and deaths were uncommon in both groups. CONCLUSIONS:Following a third dose of an mRNA-based vaccine, infliximab was associated with attenuated serological responses and more SARS-CoV-2 breakthrough infection and reinfection which were not predicted by the magnitude of anti-S RBD responses, indicative of vaccine escape by the Omicron variant. TRIAL REGISTRATION NUMBER:ISRCTN45176516.

Background Whole-genome sequencing has been used successfully to provide additional information for transmission pathways in infectious spread. We report and interpret genomic sequencing results in clinical context from a large outbreak of COVID-19 with 46 cases across staff and patients in a community hospital in the UK. Methods Following multiple symptomatic cases within a two-week period, all staff and patients were screened by RT-PCR and staff subsequently had serology tests. Findings Thirty staff (25%) and 16 patients (62%) tested positive for COVID-19. Genomic sequencing data showed significant overlap of viral haplotypes in staff who had overlapping shift patterns. Patient haplotypes were more distinct from each other but had overlap with staff haplotypes. Interpretation This study includes clinical and genomic epidemiological detail that demonstrates the value of a combined approach. Viral genetic sequencing has identified that staff transmission of COVID-19 was important in this community hospital outbreak.

With the rapid increase of aquaculture contributing to sustainable food security, comes the need to better understand seafood associated diseases. One of the major aquatic bacterial genera responsible for human infections from seafood isVibrio, especially from oysters. Currently,in vivostudy of bacterial interactions within oysters is limited by the inability to promote high-level uptake of bacteria by oysters. This study has therefore evolved current natural marine snow protocols to generate 'artificial' marine snow, into which bacteria can be incorporated to facilitate extensive uptake by oysters. This presents an adaptable model for bacterial study within filter-feeding shellfish. Using this model, we demonstrate for the first time the antibacterial activity ofVibrio vulnificusType 6 secretion systemsin vivo, revealing an important role for the T6SS inV.vulnificusecology.

The Coronavirus Disease 2019 (COVID-19) Genomics UK Consortium (COG-UK) was launched in March, 2020, with £20 million support from UK Research and Innovation, the UK Department of Health and Social Care, and Wellcome Trust. The goal of this consortium is to sequence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for up to 230 000 patients, health-care workers, and other essential workers in the UK with COVID-19, which will help to enable the tracking of SARS-CoV-2 transmission, identify viral mutations, and integrate with health data to assess how the viral genome interacts with cofactors and consequences of COVID-19. Results from this initiative are guiding decision makers, including the weekly reports to the UK Scientific Advisory Group for Emergencies (SAGE). This initiative is the first time that large-scale genomic epidemiology has been used to guide and inform the public health response to a pandemic in the UK, setting the stage for genomics to serve as a core tool for outbreak tracking in future pandemics. COG-UK builds on the UK's strengths in pathogen genomics, population health sciences, and health informatics. It benefits from a large and well equipped network of specialist academic and research facilities working in close collaboration with the UK's public health agencies and the National Health Service (NHS). Partners in COG-UK at the time of writing are shown in the appendix (p 4). As COG-UK's example could help to inform other countries seeking to rapidly develop and scale-up national sequencing capacity and a joined-up health information system, we describe six key features of our experience. COG-UK is co-ordinated from an integrated hub (based between the University of Cambridge [Cambridge, UK] and Wellcome Sanger Institute [Cambridge, UK]), with sample collection and sequencing taking place at multiple organisations across the country. This decentralised model enables rapid sequencing and prioritisation at the point of need, while supporting equitable national access. The core of COG-UK is formed around a network of regional sequencing centres in UK academic institutions and UK public health agencies. Sequencing in regional sequencing centres is close to real time, with a 24–48 h turnaround time, with data interpreted and used locally. In addition, the Wellcome Sanger Institute provides a high volume national sequencing capacity using high throughput, cost efficient viral sequencing for UK hospitals that do not have sequencing capabilities (ie, contributing partners), the national testing centres for key workers, and overspill from the regional sequencing centres. To help prioritise finite sequencing resources, COG-UK has developed a sampling strategy to concurrently enable broad population-level analyses, targeted analyses of specific populations, and freedom to tackle local priorities. This strategy aims to maximise insight while minimising demands on stretched local facilities. To maximise speed and inclusiveness, COG-UK adopts a range of technical approaches (ie, amplicons, baits, and metagenomics) and technologies (ie, single molecule vs next generation sequencing approaches), enabling centres to build on existing pipelines and expertise. Several centres have adopted a tiling amplicon sequencing approach developed by the ARTIC network, which is run on multiple platforms to help achieve the very high sample throughput required. Tight linkage to diagnostic and public health laboratories minimises transport and analysis delays. The system needs to be continuous and rapid, with a target of 48 h from sample collection to analysis. Sequence data are uploaded to the Cloud Infrastructure for Microbial Bioinformatics (MRC-CLIMB) server;1Connor TR Loman NJ Thompson S et al.CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online resource for the medical microbiology community.Microb Genom. 2016; 2e000086PubMed Google Scholar a centralised, replicated environment for data storage and analysis. MRC-CLIMB provides a ready-made starting point for the computational analysis, with the option to integrate with other resources within the UK or scale into the commercial cloud. A standardised lineage assignment to enable national and international comparison has been developed,2Rambaut A Holmes EC Hill V et al.A dynamic nomenclature proposal for SARS-CoV-2 to assist genomic epidemiology.bioRxiv. 2020; (published online April 19.)DOI: 10.1101/2020.04.17.046086Google Scholar which are linked to data and released interactively via Microreact.3Argimón S Abudahab K Goater RJE et al.Microreact: visualizing and sharing data for genomic epidemiology and phylogeography.Microb Genom. 2016; 2e000093Crossref PubMed Scopus (363) Google Scholar Sequence data are made open access through release into the European Nucleotide Archive through the European Bioinformatics Institute and the Global Initiative on Sharing All Influenza Data. To enhance the value of sequence data, we are creating an integrated dataset connecting viral genome data with multidimensional patient data from clinical, epidemiological, and other sources. By accessing existing NHS e-health records and related sources, the goal is to avoid adding to the burden on busy NHS staff. Each week a sequence data cutoff is applied across the consortium to give a defined dataset for weekly analysis to report to SAGE, initially focusing on: (1) local transmission versus imported cases,4Baillie GJ Galiano M Agapow P-M et al.Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.J Virol. 2012; 86: 11-18Crossref PubMed Scopus (89) Google Scholar (2) rates of epidemic growth,5Fraser C Donnelly CA Cauchemez S et al.Pandemic potential of a strain of influenza a (h1n1): early findings.Science. 2009; 324: 1557-1561Crossref PubMed Scopus (1561) Google Scholar, 6Volz EM Kosakovsky Pond SL Ward MJ Leigh Brown AJ Frost SD Phylodynamics of infectious disease epidemics.Genetics. 2009; 183: 1421-1430Crossref PubMed Scopus (154) Google Scholar (3) reconstructing spatial movement, (4) chains of transmission,7Houlihan CF Frampton D Ferns RB et al.Use of whole-genome sequencing in the investigation of a nosocomial influenza virus outbreak.J Infect Dis. 2018; 218: 1485-1489Crossref PubMed Scopus (41) Google Scholar (5) observed genetic changes, and (6) identification of genomic changes potentially affecting common diagnostic tests or direct (eg, chemotherapeutics) or indirect therapies. Our website will provide a publication strategy, including open access publications, bespoke local and regional analyses, and data summaries suitable for the general public. In only 4 weeks, over 7000 genomes have been sequenced by COG-UK, the largest number of any country to date. We are committed to open and global collaboration. Reciprocity is crucial to genomic epidemiological approaches; the use of our data will be maximised if other countries adopt similar approaches. We declare no competing interests. COG-UK is supported by the funding from the UK Research and Innovation (UKRI), Medical Research Council, and the National Institute of Health Research and Wellcome, administered by UKRI. Download .pdf (.18 MB) Help with pdf files Supplementary appendix

Clostridioides difficile is the primary cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease resulting in a significant fatality rate. Colonization of the gut is critical for C. difficile pathogenesis. The bacterial molecules essential for efficient colonization therefore offer great potential as vaccine candidates. Here we present findings demonstrating that the C. difficile immunogenic lipoprotein CD0873 plays a critical role in pathogen success in vivo. We found that in a dixenic colonization model, a CD0873-positive strain of C. difficile significantly outcompeted a CD0873-negative strain. Immunization of mice with recombinant CD0873 prevented long-term gut colonization and was correlated with a strong secretory IgA immune response. We further present high-resolution crystal structures of CD0873, at 1.35?2.50 ? resolutions, offering a first view of the ligand-binding pocket of CD0873 and provide evidence that this lipoprotein adhesin is part of a tyrosine import system, an amino acid key in C. difficile infection. These findings suggest that CD0873 could serve as an effective component in a vaccine against C. difficile.

ABSTRACTClinical scorecards of risk factors associated with disease severity or mortality outcome are used by clinicians to make treatment decisions and optimize resources. This study develops an automated tool or framework based on evolutionary algorithms for the derivation of scorecards from clinical data. The techniques employed are based on the NSGA-II Multi-objective Optimization Genetic Algorithm (GA) which optimizes the Pareto-front of two clinically-relevant scorecard objectives, size and accuracy. Three automated methods are presented which improve on previous manually derived scorecards. The first is a hybrid algorithm which uses the GA for feature selection and a decision tree for scorecard generation. In the second, the GA generates the full scorecard. The third is an extended full scoring system in which the GA also generates the scorecard scores. In this system combinations of features and thresholds for each scorecard point are selected by the algorithm and the evolutionary process is used to discover near-optimal Pareto-fronts of scorecards for exploration by expert decision makers. This is shown to produce scorecards that improve upon a human derived example for C.Difficile, an important infection found globally in communities and hospitals, although the methods described are applicable to any disease where the required data is available.

Diabetic foot infection is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. Recent figures published by Public Health England show that there are more than 163 diabetes related amputations in England every week. This study investigates the effect of antibiotic loaded calcium sulfate (Stimulan® Rapid Cure) beads on in-vitro bacterial growth from tissue taken from diabetic foot infections. Patients were recruited from the Macleod Diabetes and Endocrine Centre at the Royal Devon and Exeter Hospital. Inclusion in the study was based on clinical recognition of an infected foot ulcer requiring wound debridement. Debrided tissue was homogenised and 50 µl spread over the surface of Columbia blood agar and fastidious anaerobe agar. Three replicate calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar. Each bead contained approximately 3.4 mg and 1.6 mg of vancomycin and gentamicin respectively. Plates were incubated aerobically or anaerobically as appropriate. Zones of inhibition were recorded at 1 and 4 days. Calcium sulfate beads containing vancomycin and gentamicin were able to inhibit bacterial growth in all tissue homogenates tested with zone diameters ranging from 16 to 40 mm. Local release of antibiotics could have the benefit of achieving high local concentrations within poorly vascularised tissue which may inhibit bacterial growth at the wound site. By improving treatment of diabetic foot infections, it may be possible to prevent amputation, maintain mobility and conserve quality of life.

Vibrio vulnificus is a bacterium responsible for severe gastroenteritis, sepsis and wound infections. Gastroenteritis and sepsis are commonly associated with the consumption of raw oysters, whereas wound infection is often associated with the handling of contaminated fish. Although classical virulence factors of this emerging pathogen are well characterised, there remains a paucity of knowledge regarding the general biology of this species. To investigate the presence of previously unreported virulence factors, we applied whole genome sequencing to a panel of ten V. vulnificus strains with varying virulence potentials. This identified two novel type 6 secretion systems (T6SSs), systems that are known to have a role in bacterial virulence and population dynamics. By utilising a range of molecular techniques and assays we have demonstrated the functionality of one of these T6SSs. Furthermore, we have shown that this system is subject to thermoregulation and is negatively regulated by increasing salinity concentrations. This secretion system was also shown to be involved in the killing of V. vulnificus strains that did not possess this system and a model is proposed as to how this interaction may contribute to population dynamics within V. vulnificus strains. In addition to this intra-species killing, this system also contributes to the killing of inter bacterial species and may have a role in the general composition of Vibrio species in the environment.

BACKGROUND:The aim of this study was to characterize the elution of four antibiotics from pharmaceutical-grade calcium sulfate beads and show that the eluted antibiotics retained efficacy.METHODS:Calcium sulfate was combined with gentamicin, tobramycin, vancomycin, or rifampicin (ratio: 20 g of calcium sulfate, to 240 mg, 500 mg, 900 mg, and 600 mg of antibiotic, respectively). Three grams of beads were immersed in 4 mL of sterile phosphate-buffered saline (PBS) at 37°C. At each time point (4, 8, 24 h; 2, 7, 14, 28, 42 d), eluates were removed for analysis by liquid chromatography-mass spectrometry. The antimicrobial efficacy of antibiotics combined with calcium sulfate beads after 42 d was tested by a modified Kirby-Bauer disc diffusion assay.RESULTS:All samples showed a generally exponential decay in the eluted antibiotic concentration. At the first time point, both gentamicin and tobramycin had eluted to a peak concentration of approximately 10,000 mcg/mL. For rifampicin, the peak concentration occurred at 24 h, whereas for vancomycin, it occurred at 48 h. The eluted concentrations exceeded the minimum inhibitory concentration for common periprosthetic joint infection pathogens for the entire span of the 42 study days. Mass spectrometry confirmed all antibiotics were unchanged when eluted from the calcium sulfate carrier. Antimicrobial efficacy was unaltered after 42 d in combination with calcium sulfate at 37°C.CONCLUSIONS:Pharmaceutical-grade calcium sulfate has the potential for targeted local release of tobramycin, gentamicin, vancomycin, and rifampicin over a clinically meaningful time period.

Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.

Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

Clostridium difficile infection poses a significant healthcare burden. However, the derivation of a simple, evidence based prediction rule to assist patient management has not yet been described.

We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the structure, we applied a recently developed method for epitope design that combines computational epitope predictions with in vitro mapping experiments and successfully identified a consensus sequence within the antigen that, when engineered as a synthetic peptide, was selectively immunorecognized to the same extent as the recombinant protein in sera from melioidosis-affected subjects. Antibodies raised against the consensus peptide were successfully tested in opsonization bacterial killing experiments and antibody-dependent agglutination tests of B. pseudomallei. Our strategy represents a step in the development of immunodiagnostics, in the production of specific antibodies and in the optimization of antigens for vaccine development, starting from structural and physicochemical principles.