The Citizen Phage Library: Rapid Isolation of Phages for the Treatment of Antibiotic Resistant Infections in the UK.

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.

Exploring the Use of Pictograms in Privacy Agreements to Facilitate Communication Between Users and Data Collecting Entities: Randomized Controlled Trial.

BACKGROUND: Privacy agreements can foster trust between users and data collecting entities by reducing the fear of data sharing. Users typically identify concerns with their data privacy settings, but due to the complexity and length of privacy agreements, users opt to quickly consent and agree to the terms without fully understanding them. OBJECTIVE: This study explores the use of pictograms as potential elements to assist in improving the transparency and explanation of privacy agreements. METHODS: During the development of the pictograms, the Double Diamond design process was applied for 3 instances of user interactions and 3 iterations of pictograms. The testing was done by performing a comparative study between a control group, which received no pictograms, and an experimental group, which received pictograms. The pictograms were individually tested to assess their efficacy by using an estimated comprehension of information symbols test. RESULTS: A total of 57 participants were recruited for the pictogram evaluation phase. With the addition of pictograms, the overall understanding improved by 13% (P=.001), and the average time spent answering the questions decreased by 57.33 seconds. A 9% decrease in perceived user frustration was also reported by users, but the difference was not significant (χ24=4.80; P=.31). Additionally, none of the pictograms passed the estimated comprehension of information symbols test, with 7 being discarded immediately and 5 requiring further testing to assess their efficacy. CONCLUSIONS: The addition of pictograms appeared to improve users' understanding of the privacy agreements, despite the pictograms needing further changes to be more understandable. This proves that with the aid of pictographic images, it is possible to make privacy agreements more accessible, thereby allowing trust and open communication to be fostered between users and data collecting entities. TRIAL REGISTRATION: ClinicalTrials.gov NCT05631210; https://clinicaltrials.gov/ct2/show/NCT05631210.

Vaccine escape, increased breakthrough and reinfection in infliximab-treated patients with IBD during the Omicron wave of the SARS-CoV-2 pandemic.

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.

In vitro efficacy of antibiotic loaded calcium sulfate beads (Stimulan Rapid Cure) against polymicrobial communities and individual bacterial strains derived from diabetic foot infections.

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.

A universal oyster infection model demonstrates that Vibrio vulnificus Type 6 secretion systems have antibacterial activity in vivo.

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 is Vibrio, especially from oysters. Currently, in vivo study 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 of Vibrio vulnificus Type 6 secretion systems in vivo, revealing an important role for the T6SS in V. vulnificus ecology.

Molecular features of lipoprotein CD0873: A potential vaccine against the human pathogen Clostridioides difficile.

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 Å 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.

Vibrio vulnificus Type 6 Secretion System 1 Contains Anti-Bacterial Properties.

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.

Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation.

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 pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

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.

Local release of antibiotics for surgical site infection management using high-purity calcium sulfate: an in vitro elution study.

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.

Gene and protein expression in response to different growth temperatures and oxygen availability in Burkholderia thailandensis.

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.

Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

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.

Exploiting the Burkholderia pseudomallei acute phase antigen BPSL2765 for structure-based epitope discovery/design in structural vaccinology.

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.

Derivation and validation of a simple, accurate and robust prediction rule for risk of mortality in patients with Clostridium difficile infection.

BACKGROUND: 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. METHOD: Univariate, multivariate and decision tree procedures were used to deduce a prediction rule from over 186 variables; retrospectively collated from clinical data for 213 patients. The resulting prediction rule was validated on independent data from a cohort of 158 patients described by Bhangu et al. (Colorectal Disease, 12(3):241-246, 2010). RESULTS: Serum albumin levels (g/L) (P = 0.001), respiratory rate (resps /min) (P = 0.002), C-reactive protein (mg/L) (P = 0.034) and white cell count (mcL) (P = 0.049) were predictors of all-cause mortality. Threshold levels of serum albumin ≤ 24.5 g/L, C- reactive protein >228 mg/L, respiratory rate >17 resps/min and white cell count >12 × 10(3) mcL were associated with an increased risk of all-cause mortality. A simple four variable prediction rule was devised based on these threshold levels and when tested on the initial data, yield an area under the curve score of 0.754 (P < 0.001) using receiver operating characteristics. The prediction rule was then evaluated using independent data, and yield an area under the curve score of 0.653 (P = 0.001). CONCLUSIONS: Four easily measurable clinical variables can be used to assess the risk of mortality of patients with Clostridium difficile infection and remains robust with respect to independent data.

Emergence and global spread of epidemic healthcare-associated Clostridium difficile.

Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance-conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells.

Host gene products required for mediating the action of toxins are potential targets for reversing or controlling their pathogenic impact following exposure. To identify such targets libraries of insertional gene-trap mutations generated with a PiggyBac transposon in Blm-deficient embryonic stem cells were exposed to the plant toxin, ricin. Resistant clones were isolated and genetically characterised and one was found to be a homozygous mutant of the mannosidase 2, alpha 1 (Man2α1) locus with a matching defect in the homologous allele. The causality of the molecular lesion was confirmed by removal of the transposon following expression of PB-transposase. Comparative glycomic and lectin binding analysis of the Man2α1 (-/-) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal ß-galactose moieties, potential target receptors for ricin. Furthermore, naïve ES cells treated with inhibitors of the N-linked glycosylation pathway at the mannosidase 2, alpha 1 step exhibited either full or partial resistance to ricin. Therefore, we conclusively identified mannosidase 2, alpha 1 deficiency to be associated with ricin resistance.

Glycosylation of DsbA in Francisella tularensis subsp. tularensis.

In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis.

Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide.