Saudi Cultural Missions Theses & Dissertations
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Item 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.12 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.14 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.12 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.29 0Item Restricted UNRAVELING THE COMPLEXITY OF CDEC EXPRESSION DURING CLOSTRIDIOIDES DIFFICILE SPORULATION: INSIGHTS INTO THE ROLE OF SIGMA FACTORS AND NON-CODING RNA ON CDEC EXPRESSION(Texas A&M University, 2024) Alharbi, Areej; Paredes-Sabja, DanielGram-positive, obligatory anaerobic Clostridioides difficile (C. difficile) is a spore- forming, obligate anaerobic bacterium that causes diarrhea and other healthcare-associated illnesses globally. This study aimed to identify the sigma factor boxes in the promoter region of cdeC that affect the early and late stages of C. difficile sporulation. Therefore, we first identified the sigma factors' positions in the promoter region. Then, we constructed transcriptional fusions containing a different deletion of these sigma factors and introduced them into the C. difficile R20291CM210 WT strain. However, no detectable fluorescence was observed due to lack of sporulation in the C. difficile R20291CM210. Both increasing the time of incubation and increasing thiamphenicol concentrations failed to induce sporulation for R20291CM210. After that, we introduced all transcriptional fusions into the C. difficile R20291CM196 WT strain. We found out that only P∆ncRNA-cdeC-mScarlett transcriptional fusion that contains the promoter region with a deletion of the ncRNA shows a modest fluorescence signal compared to other fusions. These results suggest that ncRNA located in the promoter region of cdeC may have an activity for cdeC expression during sporulation.32 0Item Restricted The role of commensal bacteria in the maintenance of the tight junction skin barrier(Saudi Digital Library, 2023) Aldehalan, Faye; Catherine, O'neillThe skin is a critical barrier that prevents water and heat loss from the body and prevents ingress of toxins and pathogens. This barrier comprises the physical (stratum corneum and tight junctions (TJs)), chemical and immune components. Recent studies have shown that the skin microbiota also plays a role in epidermal barrier function by preventing infection by pathogenic bacteria and enhancing skin immune responses. Some recent studies have also suggested that the physical barrier can be regulated by bacterial metabolites such as butyrate, from the fermentation of glycerol. However, to date most studies have been conducted in mouse models using consortia of bacteria and few studies have looked at individual bacteria and their effects on the barrier. The aim of this doctoral thesis was to identify human skin bacterial isolates that have the potential to regulate TJ barrier function possibly via production of butyrate and other metabolites. Skin swabs were collected from five healthy participants to create a biobank of skin commensal bacteria using culture-based methods. The isolated species were used in screening assays of TJ function in primary normal human epidermal keratinocytes (NHEKs). One of the enhancers of the intestinal barrier is butyrate, a bacterial metabolite from fermentation of carbohydrates. Similar mechanisms may also exist in the skin since some skin commensal bacteria have been shown to produce butyrate from the fermentation of glycerol. A significant increase in transepithelial electrical resistance (TEER) was observed in NHEKs treated with 4 mM butyrate for 24 h compared to untreated cells. However, treatment with lower concentrations (2 and 0.5 mM) induced no change in TEER. Staphylococcal isolates were able to ferment glycerol as a source of carbohydrate, but the concentrations of butyrate produced were low. In keeping with this, the glycerol-fermented supernatants of all of the tested species (S. epidermidis, S. hominis, S. capitis S. lugdunensis, and S. caprae) had no impact on TJ barrier function in NHEKs. The effect of cell-free supernatants of different skin commensal isolates grown in tryptic soy broth (to maximise the production of metabolites), on TJ integrity were investigated in NHEKs. The supernatants from S. hominis, S. aureus and M. luteus significantly increased the TJ barrier integrity as measured by TEER. However, the supernatants of S. epidermidis, S. capitis S. lugdunensis, and S. caprae had no impact on TJs. Metabolite identification by mass spectrometry showed that all the species produced multiple metabolites, with many in common between the different isolates. However, tryptophol, a known enhancer of the intestinal TJ barrier was detected only in the supernatants of the TJ barrier-enhancing species. There was almost zero production by the ineffective species. Further investigation sought to determine whether this was a general effect of a certain species, or whether there were any strain-specific effects. Therefore, other isolates of S. hominis were investigated for their ability to increase TJ function. Only specific isolates of S. hominis could regulate TJ function in NHEKs and this correlated with the production of tryptophol. Furthermore, an isolate that was unable to produce tryptophol or modify TJ function, was sequenced and found to contain mutations in a gene for tryptophol production. This study shows for the first time that specific bacteria within the skin microbiota can produce metabolites with the capacity to regulate human keratinocytes tight junction function. The identification of such activities could be utilised in the development of therapeutic approaches to different skin conditions where the barrier is known to be aberrant.10 0Item Restricted The Etiology of Peri-implantitis: Microbiological Profile Within and Around Dental Implants and the Associated Human Immune Response(2023) Kensara, Anmar; Masri, RadiObjectives: To characterize the microbiome composition within and around dental implants of peri-implantitis subjects and within and around healthy implants using 16S rRNA gene sequencing, and to profile salivary inflammatory mediators associated with peri-implantitis compared to healthy controls from the same subjects. Methods: A total of 24 subjects (peri-implantitis n=14, healthy n=10) were enrolled in the study. From the 24 subjects, 24 endosseous implants from affected (peri-implantitis) and 14 healthy controls were included in this cross-sectional study. Samples for microbiological analysis were obtained from the internal surfaces of dental implants and peri-implant sulcus using sterile paper points. DNA was extracted and 16S rRNA gene was amplified using universal primers targeting the V3-V4 regions. Amplicons were sequenced using Illumina MiSeq platform. Alpha and beta diversity, core microbiome, and taxa differential abundance were assessed. Saliva was collected from the same subjects for immunology-based assays. Salivary inflammatory mediators in peri-implantitis and healthy implant subjects were profiled using antibody arrays. Results: A significant increase in microbial diversity was observed in the internal implant surface of healthy implants compared with the internal surfaces of peri-implantitis (Shannon P= 0.02), and no significant differences in microbial diversity between healthy implants sulci and peri-implantitis pockets (Shannon P= 0.82). Bacterial community structure was significantly different within implant in both healthy and peri-implantitis groups (P= 0.012) but not significantly different around implants in both healthy and peri-implantitis (P= 0.18). Enterococci is the predominant bacteria within peri-implantitis (LD >2.0, P< 0.05). Abundant species in peri-implantitis were C. leadbetteri, T. maltophilum, Peptostreptococcus, Neisseria, P. gingivalis, and P. endodontali, L. lactis and F. alocis (P < 0.05). Gram-positive bacteria such as S. salivaris, P. melaninogenica, L. wadei, and Actinomyces spp were more abundant in the peri-implant healthy sulcus. Around 48% of detected bacteria were cultivable in general media. In addition, out of 105 analytes examined in saliva, we found that 29 mediators were upregulated in subjects with peri-implantitis (P < 0.001). Conclusions: Our results indicate that microbial colonization of the internal implant surface may act as a major contributor to the etiology of peri-implant disease. Multiple inflammatory mediators were significantly elevated in the saliva of peri-implantitis patients compared to healthy implant patients.30 0