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 A Treatment for Clostridioides difficile Based on Cell Wall Lysins Isolated from C. difficile Specific Bacteriophages(Cardiff University, 2024-04) Alyahya, Khalid Abdullah; Baillie, Les; Heard, Charles; Mehellou, YoucefClostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. While the vegetative form of the pathogen is susceptible to antibiotic treatment, its ability to persist in the gut as antibiotic-resistant spores leads to reinfection in cases where protective microflora is not reestablished. Utilizing recombinant endolysins as treatment of CDI is promising because of their activity against antibiotic and bacteriophage resistant strains. In this study, the recombinantly expressed endolysin LysCD6356 and its enzymically active domain (EAD) were shown to lyse the vegetative forms of a panel of clinical isolates of C. difficile from the UK, including hypervirulent 027 ribotype strains. While these results were promising endolysins, like antibiotics and bacteriophages, endolysins have no effect against the spore form of the pathogen, which is responsible for recurrent infections following successful treatment. To address this issue, a combination of germinants and a bactericidal agent were employed to target the more sensitive form of the pathogen. In this study, exposing of C. difficile spores to germinants followed by endolysin several hours later proved to be the most effective approach suggesting a promising approach to treat relapsing CDI. Additionally, the binding of LysCD6356 and its EAD to the spore and vegetative forms of C. difficile would leave the endolysin ideally placed to target the emerging vegetative cells. This also raise the possibility of developing a diagnostic tool that could be used to detect the pathogen. Bioinformatic studies revealed a significant similarity between LysCD6356 and its EAD to CD27L and CD271-179, respectively, suggesting a possibility of sharing common motifs. However, there was no similarity of LysCD6356 and its EAD with previously published spore binding domains, suggesting the presence of a new spore binding region. An attempt to investigate the binding of endolysins to spore surface proteins, CdeC and CdeM, was unsuccessful possibly due to deficiencies in the experimental approach or the fact that the endolysins recognize other spore surface targets. The combination of our strategy of targeting spores with more traditional approach, such as fecal microbiota transplant, could provide more efficient treatment of relapsing CDI.25 0Item Restricted MazF Toxin and Its Contribution to Phage Resistance in Staphylococcus aureus.(Saudi Digital Library, 2023-12-05) Alsulami, Raed; Xia, GuoqingMethicillin-resistant Staphylococcus aureus (MRSA) is one of the most concerning pathogens, especially in healthcare settings with significant clinical and economic burdens. In recent years, the decline in antibiotic development has highlighted the need for alternative treatments. Consequently, phage therapy offers a promising alternative to combat MRSA infections with specific targeting and notable efficacy. However, to achieve successful clinical application of phage therapy, it is crucial to study mechanisms that drive phage resistance. It has been reported that in some gram-negative bacteria, toxin-antitoxin (TA) systems play a role in phage resistance. This project aimed to study the endoribonuclease MazF toxin of the type II TA system and its contribution to phage resistance in S. aureus through different phenotypic and molecular tests. Initially, to assess MazF antiphage activity range, we evaluated the plating efficiency of Staphylococcal phage serogroups D, A and F on S. aureus JE2 mutant with a transposon insertion in mazF. The results showed that mazF mutation leads to a significant increase in susceptibility to infection by all tested phages by at least 2.1-fold compared to the wild-type. This susceptibility phenotype was also observed in mazF-transduced S. aureus COL and MW2 strains, demonstrating significantly more susceptibility to phage infection than their wild-type strains. Moreover, growth curve analysis during phage infection showed that the growth pattern of the mazF mutant strain was notably worse over time than the wild-type strain, further suggesting MazF phage resistance role. Furthermore, mazF expression post-phage infection was evaluated using RT-qPCR. The relative gene expression analysis indicated significant upregulation of mazF in response to phage infection when compared to untreated control. Our findings suggest a crucial role of MazF toxin in phage resistance in S. aureus. Also, it could point to a promising direction to support the development of phage therapy against MRSA infections.28 0Item Restricted Bacteriophage and Tail associated lysins of enterococci(Saudi Digital Library, 2023-10-10) Alrafaie, Alhassan; Stafford, Graham; Shepherd, JoeyEnterococcal species especially E. faecalis and E. faecium are among the most commonly isolated pathogens that can resist several classes of antibiotics. To mitigate the issue of AMR, bacterial viruses (phages) can be used to infect and kill these pathogens. The first step in this process involves adsorption to specific host receptors followed by genome ejection into the host. To achieve this step, specific phage proteins named Tail-associated lysins (TAL) facilitate phage infection to their hosts via locally degrading bacterial layers. Therefore, we first aimed to bioinformatically investigate the TALs in 506 enterococcal phage and prophage genomes. Prophage genomes were identified using the PHASTER web server. The identification of TALs was carried out using Pfam, NCBI domain database and PHYRE2. Using these tools, we identified various TALS: Endopeptidase, lytic transglycosylase, Pectinesterase, New Lipoprotein C/Protein of 60-kDa (NLPC/P60) and Glycerophosphodiester phosphodiesterase (GPDP). The most common TAL identified in both phage and prophage genomes was endopeptidase followed by lytic transglycosylase. The identified TALs have different targets with endopeptidase and lytic transglycosylase targeting the bacterial peptidoglycan structure, GPDP degrading teichoic acids and pectinesterase possibly attacking enterococcal polysaccharide antigen (EPA). Interestingly, the identified NLPC/P60 proteins were correlated to the phage genomic classifications and host specificity. Phages are found almost everywhere and they have been isolated from several sources such as wastewater. Therefore, we collected and processed wastewater samples in our lab for phage isolation. To assess these samples, TEM was used to visualise various phage morphologies. Using these samples, 8 phages were isolated that E. faecalis and E. faecium strains in enrichment experiments, including clinical strains isolated from patients with diabetic foot ulcers. Following isolation, phage morphology was assessed via TEM with sipho-, myo- and podovirus phages observed. Since phages are very specific in terms of infecting bacteria, we have tested the ability of the isolated phages to infect 36 E. faecalis and E. faecium strains. A genomic analysis was then performed which revealed 5 novel strictly lytic phages. Since phiSHEF13 showed the broadest host range, we aimed to further characterise this phage. An experiment to assess phage host receptors was carried out which revealed that the variable region (epaV) of the E. faecalis strain V583 is essential for phiSHEF13 successful infection. As three different phages were isolated using the E. faecium E1071 strains, killing assays were conducted which revealed quick bacterial inhibition at different MOI. Phage-resistant mutants were also observed and investigated. The last chapter in this thesis is about assessing TALs In vitro by cloning and expressing candidate proteins. First, we selected one example from each identified lysin (a total of five proteins) and the candidate proteins were subcloned into vectors possessing His or GST tags and expressed using BL21(DE3) or C41(DE3). After expression, two TAL proteins namely pectinesterase and NLPC/P60 were successfully expressed and purified. To conclude, the isolation of novel lytic phages would facilitate phage therapy targeting antibiotic-resistant enterococci. Moreover, the identification of various lytic proteins would also provide alternative use of phage to tackle antibiotic-resistant bacteria.19 0