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 Clinical Investigation of the Impact of Endodontic Disinfection on the Bacteriome of Root Canal Infection Using Next-Generation Sequencing on the Illumina MiSeq Platform(University of Maryland, Baltimore, 2024-07-01) Alquria, Theeb; Martinho, FredericoThe primary cause of root canal infection is bacteria and their by-products, making disinfection of the root canal system a key goal in endodontic therapy. However, the complex anatomy of root canal systems, particularly the isthmus and its ramifications, poses challenges for effective disinfection. Currently, no disinfection protocol can eliminate all bacterial contents from root canal infections, driving the ongoing search for an optimal disinfection approach. Recently, next-generation sequencing (NGS), particularly the Illumina MiSeq platform, has been widely explored in endodontic infections due to its low sequencing error rates, cost-effectiveness, and high-quality reads. Leveraging advanced sequencing techniques to reveal the bacteriome of root canal infections and assess the impact of current disinfection methods could enable the development of more targeted and effective disinfection protocols. This dissertation presents an interventional clinical study aiming to investigate the diversity and composition of the bacteriome in primary endodontic infection (PEI) with apical periodontitis (AP) and evaluate the impact of root canal disinfection on the endodontic bacteriome using NGS on the Illumina MiSeq Platform. First, we characterized the bacteriome in PEI with AP, identified core and rare bacteriome species, and analyzed community diversity metrics using the Illumina MiSeq platform. Our results showed that Bacteroidetes, Firmicutes, Synergistetes, Fusobacteria, and Actinobacteria were the most abundant bacterial phyla. We identified 113 genera and 215 species. Analysis revealed differences in abundant taxa among distinct age, gender, symptomatology, and lesion size groups. These findings suggest that the bacteriome in PEI with AP is complex and has high microbial heterogeneity among patients. Moreover, age, gender, symptomatology, and lesion size might play a role in the abundant taxa present in PEI with AP. Second, we determined quantitatively and qualitatively the impact of chemomechanical preparation (CMP) using 2.5% sodium hypochlorite (NaOCl) on the bacteriome found in PEI with AP using the Illumina MiSeq platform. Despite a significant decrease in bacterial abundance, our findings demonstrated a distinct community composition and increased alpha diversity after CMP. We observed differential enrichment of specific taxa, including Stenotrophomonas_unclassified, Enterococcus_unclassified, and Actinomyces_unclassified, suggesting lower effectiveness of CMP using 2.5% NaOCl against these taxa. Findings from this dissertation highlight the complexity and heterogeneity of the bacteriome in PEI with AP, emphasizing the influence of patient-related factors on microbial diversity. The research highlighted the limited effectiveness of current endodontic disinfection protocols, specifically the use of 2.5% NaOCl, in reducing bacterial abundance while revealing limitations against certain taxa. These insights provide a foundation for developing more targeted and effective disinfection strategies, potentially leading to improved outcomes in endodontic therapy.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