Saudi Cultural Missions Theses & Dissertations
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Item Restricted Viruses of Coagulase-Negative Staphylococcus: understanding viral diversity, defence systems, and host range dynamics in the skin microbiome(Saudi Digital Library, 2029) Alsaadi, Samah Eid; Horsburgh, MalHuman skin is colonised by Staphylococcus species that have varying abundance across different body regions, with coagulase-negative staphylococcal (CoNS) species accounting for a significant proportion of the skin microbiome. Previous studies demonstrated the relative abundance of Staphylococcus species differs across skin sites and that the skin virome influences the dynamics of bacterial populations of the skin. Bacteriophages (phages) are widely present in human skin and modulate its bacterial populations, including CoNS species. This study aimed to investigate the diversity of cutaneous phages that infect major skin CoNS species, such as S. hominis, S. epidermidis, and S. capitis. Skin swabs were collected from 80 healthy volunteers at four body sites to isolate phages and tested their infection of Staphylococcus species. A total of 40 phages were isolated and genome sequenced, corresponding to six genetic clusters, with two clusters representing novel phages. Phage infection was qualitatively assessed using a wide host range of 140 strains across eight Staphylococcus species. The novel phage, named øAlsa, exhibited a greater ability to infect S. hominis, which was broadly resistant to most identified phages when compared to other CoNS species, suggesting the presence of a defence barrier that limited phage infection. Genomic analysis revealed the widespread presence of bacterial defence systems, including restriction-modification (R-M) systems, abortive infection (Abi) systems, as well as variable prophage content among species. Notably, Type IV R-M was widely present in S. hominis, which could indicate species-specific resistance that contributes to the observed phage infectivity profile. However, no definitive links between phage susceptibility and defence diversity could be determined. Biofilm formation was assessed in representative strains of S. hominis, S. epidermidis, and S. capitis, exhibiting strong biofilm formation in both phage-resistant and phage-susceptible strains, with no clear correlation between biofilm formation and phage resistance. However, Alsa phages, specifically øAlsa_2, reduced biofilm biomass, suggesting phage-associated activities that could influence receptor accessibility. Together, these findings highlight the intricate relationships between staphylococcal phages, bacterial defence systems, receptor accessibility, and biofilm formation, which collectively contribute to the populations of CoNS and their phages on human skin.9 0Item Restricted Viruses of Coagulase-Negative Staphylococcus: understanding viral diversity, defence systems, and host range dynamics in the skin microbiome(Saudi Digital Library, 2025) Alsaadi, Samah Eid; Horsburgh, MalHuman skin is colonised by Staphylococcus species that have varying abundance across different body regions, with coagulase-negative staphylococcal (CoNS) species accounting for a significant proportion of the skin microbiome. Previous studies demonstrated the relative abundance of Staphylococcus species differs across skin sites and that the skin virome influences the dynamics of bacterial populations of the skin. Bacteriophages (phages) are widely present in human skin and modulate its bacterial populations, including CoNS species. This study aimed to investigate the diversity of cutaneous phages that infect major skin CoNS species, such as S. hominis, S. epidermidis, and S. capitis. Skin swabs were collected from 80 healthy volunteers at four body sites to isolate phages and tested their infection of Staphylococcus species. A total of 40 phages were isolated and genome sequenced, corresponding to six genetic clusters, with two clusters representing novel phages. Phage infection was qualitatively assessed using a wide host range of 140 strains across eight Staphylococcus species. The novel phage, named øAlsa, exhibited a greater ability to infect S. hominis, which was broadly resistant to most identified phages when compared to other CoNS species, suggesting the presence of a defence barrier that limited phage infection. Genomic analysis revealed the widespread presence of bacterial defence systems, including restriction-modification (R-M) systems, abortive infection (Abi) systems, as well as variable prophage content among species. Notably, Type IV R-M was widely present in S. hominis, which could indicate species-specific resistance that contributes to the observed phage infectivity profile. However, no definitive links between phage susceptibility and defence diversity could be determined. Biofilm formation was assessed in representative strains of S. hominis, S. epidermidis, and S. capitis, exhibiting strong biofilm formation in both phage-resistant and phage-susceptible strains, with no clear correlation between biofilm formation and phage resistance. However, Alsa phages, specifically øAlsa_2, reduced biofilm biomass, suggesting phage-associated activities that could influence receptor accessibility. Together, these findings highlight the intricate relationships between staphylococcal phages, bacterial defence systems, receptor accessibility, and biofilm formation, which collectively contribute to the populations of CoNS and their phages on human skin.9 0Item Restricted Investigation of Novel Type III Secretion System Protein Effectors of Aeromonas veronii(Saudi Digital Library, 2025) Alfaify, Abdulkhaleg; Shaw, JonathanThe type III secretion system (T3SS) plays a crucial role in the pathogenesis of various Gram-negative bacteria, enabling them to directly inject effector proteins into host cells. This study focuses on the identification, characterization, and functional analysis of putative T3SS effector proteins (AopX, PteB, and PteA) in Aeromonas veronii. Insertional gene mutants were generated and evaluated using the Galleria mellonella larvae infection model, showing significant alterations in virulence phenotypes. Transcriptomic analysis of an exsD mutant strain provided insights into the regulation of these novel effectors and demonstrated that they were part of theT3SS regulon. The study further characterized these proteins through expressing them in both Saccharomyces cerevisiae and A549 mammalian cells. Fluorescence imaging analysis showed that these proteins had significant impacts on host cell morphology and cytoskeletal organization. Additionally, BioID proximity labelling combined with mass spectrometry proteomics analysis identified host protein interactions and key cellular pathways that influenced by these bacterial effectors. Furthermore, protein purification was optimized using various chromatography approaches and computational modelling via AlphaFold and Phyre2 was used to predict and align protein structures. Overall we found that AopX, PteB, and PteA are regulated by the T3SS regulon. AopX primarily disrupts the actin cytoskeleton in both yeast and A549 cells, most probably through interacting with Filamin A, while PteB prevents bud formation and affects cell cycle progression by interacting with Serine/Threonine phosphatases. This comprehensive investigation enhances our understanding of T3SS effector proteins in A. veronii pathogenesis and provides insights into their regulation, structural features, and specific mechanisms of host cell manipulation.28 0Item Restricted Running the Gauntlet of the Bacterial Cell Wall(Newcastle University, 2025) Alofi, Amirah; Daniel, RichardIn nature, bacteria have a variety of shapes, from simple cocci and rods to more complicated spiral or appendage structures. In their life, they need a strong exoskeleton that can maintain their shape during growth and protect them from changes in environmental conditions. This rigid structure is generally provided by the cell wall and is considered an essential component in the bacterial cell. However, the cell wall represents a complex logistical problem in terms of the controlled synthesis and degradation that is required to maintain shape, permit enlargement, and prevent lysis. Significant advances have been made in understanding wall synthesis, and in Gram-negative bacteria synthesis can be integrated with degradation. But, for Gram-positive bacteria the regulation of cell wall degradation is poorly characterised. Genetically the genes encoding the key enzymes are known and the expression of these enzymes are known to be highly regulated. However, understanding how the biochemical activity of these enzymes is restricted and regulated, presumably within the cell wall or on the outside surface of the cell is not clear. The results obtained in this laboratory have indicated that altered cell wall composition or media components modulate the level of the hydrolysis enzyme activity in some way during vegetive growth. This study aimed to extend our understanding of the autolytic systems and how the enzymatic activity may be regulated by components of the cell envelope or minor modifications of the cell wall material.20 0Item Restricted Control of Spoilage Bacteria in Lamb Meat by Using Bacteriophage(The Royal Melbourne Institute of Technology (RMIT University), 2025-08) Altakhis, Mohammed; Osborn, MarkLamb meat, known for its distinctive taste and high nutritional value, is a significant part of the human diet worldwide. However, it is susceptible to bacterial contamination, which can compromise its quality and safety. This thesis explores the bacterial community dynamics of fresh and chilled backstrap lamb meat and investigates the feasibility of using bacteriophages (viruses that infect and kill bacteria), as a novel method to control bacterial growth, including spoilage and pathogenic bacteria, in lamb meat. The research aimed to assess changes in bacterial communities, particularly specific spoilage organisms (SSO), in Modified Atmosphere Packaged (MAP) lamb meat stored at 4°C over 35 days. Methods used included 16S rRNA-gene-based sequencing, MALDI-TOF MS, and sequencing for bacterial identification. Culture-based analysis using Brain Heart Infusion (BHI) media was used to monitor changes in the bacterial population of Modified atmosphere packaging (MAP) lamb meat stored at 4°C. This comprehensive approach allowed for detailed bacterial community profiling and an understanding of bacterial stability and spoilage trajectories in stored meat products. The investigation revealed a diverse culturable bacterial community in lamb backstrap meat packaged under modified atmosphere conditions over time in chilled storage. MALDI- TOF profiling identified spoilage-associated taxa such as Pseudomonas and Acinetobacter, which thrive in refrigerated, MAP meats. Quantitative assessments of viable counts depicted an increase over time in aerobic bacterial loads and a variable yet overall increasing anaerobic population. These trends were most pronounced post-day 14, indicating significant increases in bacterial numbers present on meat. 1 Principal Coordinate Analysis (PCoA) based on Bray-Curtis, Jaccard, unweighted emperor, and weighted emperor dissimilarities was used to chart successional changes in bacterial community structure and composition based on molecular analysis of total bacterial communities. Changes in bacterial diversity was assessed using Chao1 and Shannon indices, revealing a decrease in both richness and evenness, suggesting a simplification of the communities over time. This trend was mirrored in phylogenetic diversity measures, indicating a potential loss of less dominant taxa. The findings underscore significant shifts in bacterial community structure as typified by the loss and or appearance of new species during meat storage and highlight the importance of bacterial diversity in maintaining meat quality and shelf life. High-throughput DNA sequencing data elucidated predominant and minor bacterial taxa, highlighting the dominance of bacterial groups belonging to the class Gammaproteobacteria and Firmicutes at the end of 35-day incubation period. The data also showed that the community composition changed over time as exemplified by the appearance or disappearance of new species. Additionally, the study also explored isolating bacteriophages from lamb meat as biocontrol agents. Fresh lamb backstrap meat samples were collected, packed under MAP and non-MAP conditions, and used to attempt to isolate bacteriophages active against Pseudomonas fragi, Brochothrix thermosphacta, and Carnobacterium divergens. These taxa were selected for bacteriophage assay because they were the prevalent bacterial groups at the later stages of MAP and non-MAP incubated meat samples. Optimal growth conditions for these isolates were determined with growth curve studies of bacterial cultures incubated at 25°C. The results showed optimal OD600 values of 1.0 for Pseudomonas fragi after 6 hours, 2 0.65 for Brochothrix thermosphacta after 4 hours, and 0.44 for Carnobacterium divergens after 14 hours. Isolation of bacteriophages was performed on Double layer agar plates (DAL) using two methods: Direct isolation method and Isolation of phage using a phage amplification method. The direct isolation method detected plaques only from Pseudomonas fragi DAL plates incubated at 25°C and Carnobacterium divergens DAL plates incubated at 4°C. No plaques were seen on Brochothrix thermosphacta DAL plates. The isolation of phage using the amplification method, applied to samples from days 0 to 35, detected plaques only from Brochothrix thermosphacta DAL plates incubated at 25°C. Unfortunately, none of the plaques could be re-propagated despite many attempts, indicating a need to optimize propagation methodologies. To investigate the potential use and efficacy of phage to reduce the numbers of spoilage bacteria present on meat, a commercially available phage (Pseudomonas phage vB_pfrM-S117) from a culture collection was used to investigate phage treatment of Pseudomonas fragi populations in irradiated meat samples at two time points (Day 2 and Day 7) using Tryptic Soy Agar (TSA) and Cetrimide Fucidin Nalidixic Acid (CFN) agar. On Day 2, bacteriophage treatment reduced bacterial counts by approximately 25% on TSA and 50% on CFN agar compared to samples in which phage were absent. (p < 0.05). By Day 7, the reduction increased to 70% on TSA and 57% on CFN agar. Control samples remained sterile, confirming the effectiveness of the irradiation process. These findings underscore the potential of bacteriophage therapy as a promising biocontrol strategy to managing and reduce Pseudomonas fragi in meat products. Bacteriophage-treated samples showed significant reductions in bacterial counts, highlighting the efficacy of phages in lysing bacterial cells and reducing bacterial loads. This is particularly 3 important for food safety, where controlling spoilage bacteria like Pseudomonas fragi is crucial for extending shelf life and ensuring meat quality. Additionally, bacteriophages preserve the organoleptic properties of food, unlike traditional preservation methods, by naturally controlling bacterial groups responsible for off-flavors and odors. These findings align with previous studies demonstrating the prolonged efficacy of bacteriophages in reducing bacterial populations in various food matrices, highlighting the potential of bacteriophages as effective biocontrol agents. Specifically, the key spoilage bacterial genera on lamb meat after chilled storage were identified. Subsequent assays designed to reduce bacterial population numbers using bacteriophages was carried out and for one of them, P. fragi, the application of bacteriophage (P.phage vB pfrM-S117) successfully resulted in reductions in P. fragi numbers on packaged meat. Overall, this study provides valuable insights into the potential application of bacteriophages for controlling Pseudomonas fragi in meat products. Given the significant reduction in bacterial count, the findings support the potential integration of bacteriophages into existing food safety protocols, offering a natural, targeted, and effective method for enhancing food microbiological safety. Future studies should refine bacteriophage application strategies, address potential resistance issues, and explore long-term stability and effectiveness of phage treatments in various food matrices.36 0Item Restricted Oral biofilm and host-pathogen models: a semi- systematic review and future perspectives(University Of Glasgow, 2024-08) Alshehri, Khalid; Brown, JasonAbstract Introduction: Oral biofilms, complex microbial communities found on various surfaces within the oral cavity, play a critical role in the development and progression of oral diseases such as dental caries, periodontal diseases, and mucosal infections. Understanding the formation, structure, and pathogenicity of these biofilms is essential for improving prevention and treatment strategies. Aims: This review aims to evaluate recent advancements in the development and application of in vitro multi-species oral biofilm models, with a focus on studies published between January 2019 and July 2024. The review seeks to identify gaps in current research and suggest future directions for enhancing the physiological relevance of these models. Methods: A systematic literature search was conducted in the PubMed database, following PRISMA guidelines. Studies were selected based on predefined inclusion and exclusion criteria, focusing on multi-species biofilm models in vitro. The review analyzed methodologies, findings, and limitations of the selected studies. Findings: The review identified six key studies employing various in vitro models, ranging from continuous flow systems to static models. These studies highlighted the importance of specific microbial interactions, biofilm maturation processes, and the impact of different substrates on biofilm formation. However, limitations were noted in replicating the complexity of the in vivo oral environment, particularly in capturing the dynamic conditions and microbial diversity. Discussion: While significant progress has been made in the development of in vitro biofilm models, challenges remain in creating systems that accurately mimic the oral microenvironment. Advances in microfluidic devices and 'OMICs' technologies offer promising avenues for future research. Additionally, there is a need for long-term studies that better reflect the chronic nature of biofilm-related infections. Conclusion: The development of in vitro models that closely replicate the in vivo conditions of the oral cavity is crucial for advancing our understanding of oral biofilms and their role in disease progression. Future research should focus on integrating advanced technologies and improving model complexity to enhance the predictive value of these systems for clinical applications.15 0Item Restricted Epigenetic Gene Regulation by the Type I Restriction Modification Systems(University of Leicester, 2024-04) Althari, Yasmeen; Oggioni, MarcoEpigenetic modifications mediated by type I restriction modification systems in prokaryotes have been linked to gene regulation. In Streptococcus pneumoniae, differential expression of alternative specificity subunits within the SpnIII type I restriction modification system had been shown to selectively modulate bacterial virulence. Given the presence of this system in the core genome of pneumococci, but not of the related Streptococcus mitis, SpnIII could represent a conserved phase-variable regulatory mechanism operating on a global scale. However, this phenomenon had yet to be examined at the single-gene level, and the underlying molecular mechanism remained unexplored. In this study, I utilised pneumococcal strains locked for alternative specificity subunits (no phase-variation at the locus) to validate methylation-dependent differential gene expression of several model genes using various transcriptomic and translation reporter assays. Additionally, I delved into the complexity of recombination within the spnIII locus, which is partially governed by a site-specific tyrosine recombinase whose mechanism of controlling the rate of recombination remains elusive. To investigate the regulation of this recombinase, I explored the possibility of its control by a hairpin structure in the 5-prime UTR of the gene representing a potential RNA thermosensor, considering the recognised temperature sensitivity of recombination. To provide evidence to support my hypothesis on epigenetic gene regulation, I analysed the non-phase variable prototype type I EcoKI system in Escherichia coli which revealed a similar methylation-dependent differential expression following deletion of the EcoKI methyltransferase. This discovery underscores the epigenetic impact of type I RMSs, suggesting a widespread occurrence and possibly a global relevance of this phenomenon across bacterial genera. Overall, my findings propose that methylation influences both local gene topology and global genome architecture, thus playing a crucial role in methylation-mediated regulation. This mechanism involves the interplay between methylation and DNA-binding proteins, which collectively shape the overall genome architecture and transcriptional landscape.14 0Item Restricted Legionella pneumophila Infection and The Host Unfolded Protein Response(Monash University, 2024) Alshareef, Manal Hashim; Hartland, Elizabeth; McCaffrey, KathleenLegionella pneumophila is a Gram-negative bacterium that survives in the environment by replicating within free-living amoebae. When transmitted to humans through contaminated aerosols, the pathogen infects phagocytic immune cells within the lung, such as macrophages and monocytes, to cause disease. This respiratory disease features either severe pneumonia, known as Legionnaires’ disease, or a milder infection called Pontiac fever. To survive and replicate within a eukaryotic cell, Legionella species use a type-IVB secretion system, termed Dot/Icm, to secrete >330 “effector” proteins into the host cell. Dot/Icm effectors manipulate various host processes to evade elimination by phago-lysosomal degradation and establish an intracellular replication vacuole, termed the Legionella-containing vacuole (LCV). A key feature of the LCV is its similarity to rough endoplasmic reticulum (ER) membranes raising the possibility that Legionella induces ER stress and the unfolded protein response (UPR). The UPR is a homeostatic response to ER stress that can play an important role in infection and immunity. L. pneumophila Dot/Icm effectors, including Lgt1-3, SidI, and SidL, have been previously shown to inhibit UPR signalling by blocking host cell protein synthesis. However, whether the UPR restricts L. pneumophila replication or modulates the host immune response to Legionella infection remains unknown. Here we demonstrated that L. pneumophila infection of a macrophage THP-1 cell line induces host ER stress and activates canonical UPR signalling via IRE1, PERK, and ATF6. This activation is a Dot/Icm-dependent. Using pharmacological inhibitors of UPR signalling, we also demonstrated that IRE1 RNase activity supports L. pneumophila intracellular replication in THP-1 macrophages. In contrast, pre-treatment of THP-1 macrophages with pharmacological inducers of ER stress, tunicamycin and thapsigargin prior to infection reduced L. pneumophila intracellular replication. Drug pre-treatment did not inhibit L. pneumophila growth in vitro, phagocytic uptake of the bacterium or Dot/Icm effector translocation. Although tunicamycin enhanced cell death resulting in reduced bacterial load, thapsigargin pre-treatment instead protected macrophages from L. pneumophila-induced cytotoxicity. Thapsigargin induced restriction of L. pneumophila intracellular replication relied on IRE1-kinase activity and STAT1 activation, and hence was linked to UPR-mediated immunity during ER stress. How this restriction is orchestrated needs to be further investigated. Finally, we successfully constructed a L. pneumophila mutant of strain 130b lacking Lgt1, Lgt3, SidI, and SidL, which we termed delta4. The delta4 mutant exhibited normal in vitro growth and was not different from the wild-type parent strain in terms of intracellular replication withinTHP-1 macrophages and loss of Lgt1, Lgt3, SidI, and SidL partially restored host protein synthesis and IRE1-dependent XBP1 mRNA splicing, similar to previous studies with L. pneumophila strain Philadelphia. The delta4 mutant inhibited UPR signalling in THP-1 cells early during the infection (~6 h) but not later in the replicative phase of the infection. Interestingly, wild-type L. pneumophila 130b inhibited STAT1 signalling compared to the delta4 mutant, which induced ER stress in THP-1 macrophages suggesting that Dot/Icm effectors play a role in modulating the host immune response induced by ER stress during L. pneumophila infection.17 0Item Restricted Comparison of long-read and short-read bacterial DNA sequencing(King's College London, 2024-08-30) Alqirnas, Mohammed; Carpenter, Guy; Cleaver, LeanneThis study aimed to compare long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies for characterizing the diversity and composition of in vitro oral biofilms. An oral biofilm model was established using hydroxyapatite discs to mimic the tooth surface. Saliva samples from six healthy participants were pooled and used to inoculate the discs, which underwent aerobic and anaerobic incubation phases. Biofilm formation was assessed using confocal microscopy with LIVE/DEAD staining, revealing heterogeneous growth patterns with coverage ranging from 17% to 47%. DNA extraction was carried out using the GenElute Bacterial Genomic DNA Kit, with yields showing significant variations across experiments (0-5 ng/μL). Interestingly, gel electrophoresis showed no difference in DNA fragment lengths between samples prepared for short-read and long-read sequencing. The experiment also highlighted the potential benefits of CO2-rich environments for early colonizer growth, particularly Streptococcus species. While the biofilm model was successfully established, the results underline the need for protocol optimization, particularly in DNA extraction and biofilm cultivation. This research provides insights into the complexity of oral microbiome analysis and sets the stage for future comparative studies on advanced sequencing technologies in oral microbiome. The findings also emphasize the importance of refining the in vitro experimental protocol, from biofilm cultivation to DNA sequencing and data analysis.13 0Item Restricted Comparison of long-read and short-read bacterial DNA sequencing(King's College London, 2024-08) Alqirnas, Mohammed; Carpenter, Guy; Cleaver, LeanneThis study aimed to compare long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies for characterizing the diversity and composition of in vitro oral biofilms. An oral biofilm model was established using hydroxyapatite discs to mimic the tooth surface. Saliva samples from six healthy participants were pooled and used to inoculate the discs, which underwent aerobic and anaerobic incubation phases. Biofilm formation was assessed using confocal microscopy with LIVE/DEAD staining, revealing heterogeneous growth patterns with coverage ranging from 17% to 47%. DNA extraction was carried out using the GenElute Bacterial Genomic DNA Kit, with yields showing significant variations across experiments (0-5 ng/μL). Interestingly, gel electrophoresis showed no difference in DNA fragment lengths between samples prepared for short-read and long-read sequencing. The experiment also highlighted the potential benefits of CO2-rich environments for early colonizer growth, particularly Streptococcus species. While the biofilm model was successfully established, the results underline the need for protocol optimization, particularly in DNA extraction and biofilm cultivation. This research provides insights into the complexity of oral microbiome analysis and sets the stage for future comparative studies on advanced sequencing technologies in oral microbiome. The findings also emphasize the importance of refining the in vitro experimental protocol, from biofilm cultivation to DNA sequencing and data analysis.31 0