Phage Therapy and Development of Delivery Systems for Gram-Positive Phage Endolysins

This thesis focussed on Gram positive phages and their endolysins. Here, two similar kay-like staphylococcal phages B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1) were isolated from a commercial therapeutic phage mix. Their host range was established on the Irish National MRSA bank, which included twenty one sequence types in addition relevant control strains. Based on this, distinct phages were identified and subjected to genome sequencing. The sequences were compared with the sequence of phage K (vB_SauM_K), which was also determined in this work. All three phages had a genome size of at least 139 kb, although some key differences were identified between each. The new phages B1 and JA1 possessed double stranded DNA and generally had a broader host range than phage K. A comparative genomic analysis on the phage genomes identified several (open reading frames) ORFs that were absent in the genome of phage K but present in genomes of phages B1 and JA1. One of the cloned genes from phage K was shown to encode a protein for the receptor-binding-protein and this protein was demonstrated to slightly inhibit phage adsorption. The other cloned gene encoded the phage endolysin and this peptidoglycan hydrolase were identical across all three phages and thus, the CHAPk endolysin of phage K was chosen to demonstrate the application of the endolysin for the control of staphylococci in milk. A two-log reduction in staphylococcal numbers in milk was observed. When the endolysin was introduced into a lactococcal secretion system using the pNZ8048 vector, detectable secretion was successfully demonstrated. Simultaneously, a Clostridium difficile phage endolysin, an amidase, was also cloned into the same secretion system with successful secretion also being demonstrated. In addition, this latter endolysin was also secreted from a recombinant E. coli strain, suggesting potential applications for delivery of the endolysin to the intestine from a hypothetical probiotic E. coli strain

Planetary health benefits from strengthening health workforce education on the social determinants of health.

Social inequalities are perpetuating unhealthy living and working conditions and behaviours. These causes are commonly called 'the social determinants of health'. Social inequalities are also impacting climate change and vice-versa, which, is causing profound negative impacts on planetary health. Achieving greater sustainability for human and planetary health demands that the health sector assumes a greater leadership role in addressing social inequalities. This requires equipping health and social care workers to better understand how the social determinants of health impact patients and communities. Integration of the social determinants of health into education and training will prepare the workforce to adjust clinical practice, define appropriate public health programmes and leverage cross-sector policies and mechanisms being put in place to address climate change. Educators should guide health and social workforce learners using competency-based approaches to explore critical pathways of social determinants of health, and what measurements and interventions may apply according to the structural and intermediary determinants of health and health equity. Key institutional and instructional reforms by decision-makers are also needed to ensure that the progressive integration and strengthening of education and training on the social determinants of health is delivered equitably, including by ensuring the leadership and participation of marginalized and minority groups. Training on the social determinants of health should apply broadly to three categories of health and social workforce learners, namely, those acting on global or national policies; those working in districts and communities; and those providing clinical services to individual families and patients

Comparison of Staphylococcus Phage K with Close Phage Relatives Commonly Employed in Phage Therapeutics

peer-reviewedThe increase in antibiotic resistance in pathogenic bacteria is a public health danger requiring alternative treatment options, and this has led to renewed interest in phage therapy. In this respect, we describe the distinct host ranges of Staphylococcus phage K, and two other K-like phages against 23 isolates, including 21 methicillin-resistant S. aureus (MRSA) representative sequence types representing the Irish National MRSA Reference Laboratory collection. The two K-like phages were isolated from the Fersisi therapeutic phage mix from the Tbilisi Eliava Institute, and were designated B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1). The sequence relatedness of B1 and JA1 to phage K was observed to be 95% and 94% respectively. In terms of host range on the 23 Staphylococcus isolates, B1 and JA1 infected 73.9% and 78.2% respectively, whereas K infected only 43.5%. Eleven open reading frames (ORFs) present in both phages B1 and JA1 but absent in phage K were identified by comparative genomic analysis. These ORFs were also found to be present in the genomes of phages (Team 1, vB_SauM-fRuSau02, Sb_1 and ISP) that are components of several commercial phage mixtures with reported wide host ranges. This is the first comparative study of therapeutic staphylococcal phages within the recently described genus Kayvirus

Bacteriological Analysis and Plasmid Profiles of Surfaces of Some Hospital Kitchen Equipment in Benin City, Nigeria

The surfaces of hospital kitchen equipment could be a major source of transmission of resistant pathogens to patients. Hence, the objective of this paper was to assess the bacteriological and plasmid profiles of surfaces of tables, sinks, chopping-boards, gas cookers and freezer handles hospital kitchen equipment in Benin City, Nigeria using appropriate standard microbiological techniques. Bacterial plasmids were isolated and separated using the agarose gel electrophoresis. Plasmid curing was performed using acridine orange. The antimicrobial sensitivity pattern showed that the bacterial isolates exhibited varying degree of resistance to the antibiotics. Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were highly resistant to the antibiotics, having multiple antibiotic resistances (MAR) index of 0.6. Micrococcus spp. and Klebsiella pneumoniae were the least resistant to the antibiotics. Plasmid analysis revealed the presence of single and multiple-banded plasmids with sizes ranging from 100-1000bp. After curing, all the isolates (except S. aureus, E. coli and P. aeruginosa) were sensitive to all the antibiotics, indicating a significant reduction in antibiotic resistance after curing. The results revealed that plasmids played a significant role in conferring resistance on the isolates. Regular cleaning and disinfection should be strictly observed in hospital kitchens to prevent outbreaks and spread of resistant pathogens in hospitals

The ROS Scavenger, NAC, Regulates Hepatic Vα14iNKT Cells Signaling during Fas mAb-Dependent Fulminant Liver Failure

Uncontrolled systemic activation of the immune system is an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In this study, we demonstrate that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished Fas mAb-induced FLF through suppression of Vα14iNKT cell activation, IFN-γ signaling, apoptosis and nitrotyrosine formation in liver. In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18−/− mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions. In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF. Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses

Analysis of the temporal expression of chemokines and chemokine receptors during experimental granulomatous inflammation: role and expression of MIP-1Α and MCP-1

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72640/1/sj.bjp.0704371.pd

The use of mesenchymal stromal cells in treatment of lung disorders

The therapeutic use of mesenchymal stromal cells (MSCs) represents a promising alternative clinical strategy for treating acute and chronic lung disorders. Several pre-clinical reports demonstrated that MSCs can secrete multiple paracrine factors and that their immunomodulatory properties can support endothelial and epithelial regeneration, modulate the inflammatory cascade, and protect lungs from damage. The effects of MSC transplantation into patients suffering from lung diseases should be fully evaluated through careful assessment of safety and associated risks, which is a prerequisite for translation of pre-clinical research into clinical practise. In this article we summarise the current status of pre-clinical research and review initial MSC-based clinical trials for treating lung injuries and lung disorders

Kinetics of proinflammatory cytokines after intraperitoneal injection of tribromoethanol and a tribromoethanol/xylazine combination in ICR mice

Tribromoethanol (2,2,2-tribromoethanol, TBE) is a popular injectable anesthetic agent used in mice in Korea. Our goal was to assess the risks associated with side effects (lesions) in the abdominal cavity, especially at high doses. To understand the underlying pathophysiological changes, we examined levels of cytokines through ELISA of abdominal lavage fluid and spleen collected from mice treated with low and high-dose TBE. ICR mice were anesthetized using one of the following protocols: a combination of TBE 200 mg/kg (1.25%) and xylazine 10 mg/kg; TBE 400 mg/kg (1.25%); and TBE 400 mg/kg (2.5%). Administration of high-dose TBE (400 mg/kg) increased the interleukin-1β and interleukin-6 levels in the peritoneal cavity over the short term (<1 day) compared with sham controls and low-dose TBE (200 mg/kg) groups. Cytokine expression in the low-dose TBE group was similar to the control group, whereas in the high-dose TBE group cytokine levels were higher in abdominal lavage fluid and spleen over the long term (10 days post-injection). We conclude that a combination of TBE 200 mg/kg (1.25%) and xylazine (10 mg/kg) is a safe and effective anesthetic for use in animals

Interfering with the CCL2–glycosaminoglycan axis as a potential approach to modulate neuroinflammation

Multiple Sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, involves an increased expression of monocyte chemotactic protein 1 MCP1-/CCL2. For exerting its chemotactic effects, chemokine binding to glycosaminoglycans (GAGs) is required and therefore this interaction represents a potential target for therapeutic intervention. We have designed an anti inflammatory decoy variant, Met-CCL2 (Y13AS21K Q23R), embodying increased affinity for GAGs as well as knocked out GPCR activation properties. This non-signalling dominant-negative mutant is shown here to be able to displace wild type CCL2 from GAGs by which it is supposed to interfere with the chemokine-related inflammatory response. In vivo, the anti-inflammatory properties were successfully demonstrated in a murine model of zymosan-induced peritonitis as well as in an experimental autoimmune encephalomyelitis, a model relevant for multiple sclerosis, where the compound lead to significantly reduced clinical scores due to reduction of cellular infiltrates and demyelination in spinal cord and cerebellum. These findings indicate a promising potential for future therapeutic development

Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia

SignificanceInvariant natural killer T cells (iNKT) have been found primarily patrolling inside blood vessels in the liver, where they respond to bacterial glycolipids presented by CD1d on liver macrophages. We show joint iNKT cells are localized outside of blood vessels and respond directly to the joint-homing pathogen, Borrelia burgdorferi, which causes Lyme borreliosis using multichannel spinning-disk intravital microscopy. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted its dissemination attempts into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway. These results suggest a critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier