Saudi Cultural Missions Theses & Dissertations
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Item Open Access In vitro Antibiofilm Activity of Cold Atmospheric Plasma on Enterococcus Faecalis(Univeristy of Liverpool, 2024) ALqahtani, Omar; Jarad, Fadi; Walsh, James; Longridge, Nicholas; Horsburgh, MalApical periodontitis is an infection primarily caused by bacteria that invade the dental pulp and periapical tissues, necessitating effective management to preserve tooth function and aesthetics through endodontic procedures. E. faecalis is one of the most significant bacteria implicated in endodontic treatment failure. Effective disinfection of the root canal system is crucial for reducing the bacterial load and ensuring long-term treatment success. Traditionally, NaOCl has been the gold standard for root canal disinfection due to its broad-spectrum antimicrobial properties. However, NaOCl presents several significant drawbacks, including potential safety concerns, adverse effects on dentine mechanical properties, and limited penetration into the dentinal tubules. These limitations necessitate the exploration of alternative or adjunctive disinfection methods that can offer enhanced efficacy and safety. Atmospheric plasma jet (APJ) technology has emerged as a promising alternative in various fields due to its potent antimicrobial properties. APJ has been investigated in areas such as food safety, industrial sanitation, medical sterilisation, and more recently, dentistry. The APJ generates reactive oxygen species (ROS), which are believed to play a critical role in its antimicrobial activity. Although the precise mode of action of APJ is not yet fully understood, its broad-spectrum antimicrobial effectiveness highlights its potential as a significant area of interest for researchers aiming to enhance endodontic treatment disinfection. The primary aim of this research was to investigate the antimicrobial efficacy of APJ against E. faecalis biofilms on hydroxyapatite (HA) discs. A secondary aim was to determine whether adding NaOCl 0.5% to APJ treatment would enhance its antimicrobial efficacy, thereby testing the synergistic potential of the combined treatment. In addition, phosphate-buffered saline (PBS) was used as the liquid medium and tested in two ways to evaluate its antimicrobial efficacy. The first method involved applying APJ to PBS without the HA disc sample for a determined period. The sample was then inserted into the activated PBS without further application of APJ to observe any antimicrobial activities generated by the APJ. This group was referred to as Plasma-Activated PBS (PAPBS). The second method involved exposing the sample to continuous APJ application while it was in the PBS medium. This approach aimed to enhance the disinfection process by utilising both APJ and the ROS generated in the PBS. The PBS media treated the sample with APJ during its time in PBS, resulting in a group termed Plasma-Enhanced PBS (PEPBS). The objective of comparing these two methods was to determine which technique would result in more significant bacterial reduction, thus guiding future clinical applications. The methodology involved culturing E. faecalis biofilms on HA discs in10 BHI broth under anaerobic conditions at 37°C. Biofilms were grown for different periods: 24 hours, 1 week, and 3 weeks, to represent varying stages of biofilm maturity. Treatment groups comprised a negative control PBS, a positive control (1% NaOCl), and various combinations involving APJ alone, PEPBS, PAPBS and APJ with 0.5% NaOCl. Biofilms were exposed to the antimicrobial agents for 1, 5, and 10 min. Bacterial reduction was assessed using CFU counting. For statistical analysis, a three-way ANOVA was employed to evaluate the significance of the results. The results demonstrated that while 1% NaOCl exhibited the highest reduction rate for immature biofilms, the mature biofilm showed higher resistance. In contrast, APJ showed superior performance in killing mature biofilms. APJ alone, PEPBS, and PAPBS provided significant differences compared to the control group. Notably, the combination of APJ and 0.5% NaOCl achieved 100% eradication of E. faecalis biofilms regardless of their age, highlighting a synergistic effect that significantly enhances antimicrobial efficacy, indicating the potential of APJ to improve the performance of various irrigants.16 0Item Restricted New Insights on Polymyxin Resistance in Acinetobacter baumannii(Monash University, 2024-11) Alsaadi, Yasser; Li, JianThe global health landscape is increasingly threatened by the rapid emergence of multidrug- resistant (MDR) Gram-negative ‘superbugs’, including Acinetobacter baumannii. Polymyxins, namely polymyxin B and colistin, stand as critical last-line therapeutic options. However, resistance to polymyxins continues to rise worldwide. Polymyxin resistance in A. baumannii primarily involves modifications to lipid A in the outer membrane mediated by the PmrAB two-component system. Additionally, the development of polymyxin-dependence during exposure to these antibiotics also contributes to this resistance phenotype. The overall objective of this thesis is to generate new mechanistic insights into polymyxin resistance in MDR A. baumannii. In Chapter 2, we developed an effective genome manipulation method for MDR A. baumannii, facilitating an in-frame pmrB deletion to explore the role of PmrAB in polymyxin resistance. We also reconstructed new genetic tools, crucial for phenotypic screening post homologous recombination and complementation/overexpression experiments in MDR A. baumannii. Overall, our method and genetic tools are critical for molecular research on the functions of antibiotic resistance genes in MDR A. baumannii strains. Chapter 3 investigated the role of PmrAB in the polymyxin resistance of AB5075. Deletion of pmrB resulted in increased tolerance to higher concentrations of polymyxin B (16 mg/L and 32 mg/L), without any observed modifications in lipid A. Further, a missense mutation in the gene ABUW_1353 was identified in the polymyxin-resistant AB06-32 strain. This gene is homologous to the response regulator of the QseBC system, known to be associated with polymyxin resistance in other bacterial species, but not previously reported in A. baumannii. Chapter 4 explored the effect of pmrB deletion on the A. baumannii transcriptome in the absence and presence of 4 mg/L polymyxin B. RNA sequencing showed 467 differentially expressed genes (DEGs) in AB06 (ΔpmrB) relative to AB5075. COG enrichment analysis indicated the regulatory role of PmrAB extends to various pathways, including energy production, virulence, stress responses, cell membrane structure and TCSs. Additionally, we examined the initial (1 h) and later (4 h) responses to polymyxin B, identifying a reduced number of DEGs in AB06 compared to AB5075 at both time points. Notably, the paa operon was downregulated in AB5075 post-polymyxin B exposure but upregulated in AB06 (ΔpmrB) before and unaffected afterwards. Our results are the first to implicate the PAA pathway in responding to polymyxin exposure and its potential connection to the PmrAB system in A. baumannii. In Chapter 5, we employed the mutagenesis method developed in Chapter 2 to achieve an in- frame deletion of the astA gene in the polymyxin-dependent AB5075D strain. Our results demonstrated that deletion of astA improved growth, increased polymyxin resistance, and reduced dependency. Our findings highlight the link between ast operon and arginine metabolism in A. baumannii polymyxin resistance. To the best of our knowledge, this is the first study to make a gene knockout in a polymyxin-dependent A. baumannii strain to investigate the mechanism of polymyxin dependence. In conclusion, this thesis developed a new molecular approach to investigate mechanisms underlying polymyxin resistance in A. baumannii. These mechanistic findings provide new insights into polymyxin resistance in A. baumannii and will aid in optimising the clinical use of this critical class of last-line antibiotics against MDR A. baumannii.11 0Item Restricted Functional characterization of gonococcal toxin-antitoxin systems(University of Nottingham, 2024-03) Bagabs, Salwa; Oldfield, Neil; Turner, David; Stocks, MichaelNeisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Gonorrhoea is a global health problem and one of the most common sexually transmitted infections in the world. Antibiotic resistance in N. gonorrhoeae is a growing problem, and in order to overcome this, it is necessary to develop preventative vaccines or novel treatments. Bacteria have evolved toxin-antitoxin (TA) systems to protect themselves from stressful conditions. TA systems have been linked to various functions including persister cell formation, biofilm formation, phage resistance and stabilisation of genetic elements. Previous work using the Toxin-Antitoxin DataBase (TADB) revealed the presence of three chromosomally-encoded type II TA systems (HicAB, MazEF, VapBC/FitAB) in N. gonorrhoeae strain FA1090. These were hypothesised to play an important role in the lifestyle of this human-restricted pathogen. In addition, exploitation of one or more of gonococcus TA systems could lead to novel treatment interventions. In this study, further bioinformatics analysis using PubMLST.org examined the prevalence and conservation of the TA systems across isolates. The FitAB, MazEF (and to a lesser extent the HicAB) TA systems were very highly prevalent and conserved across over 5000 N. gonorrhoeae isolates examined. To provide experimental confirmation that the predicted TA systems could influence bacterial growth, DNA fragments corresponding to fitA, fitB and both genes were amplified from N. gonorrhoeae FA1090, ligated into an arabinose-inducible expression plasmid and transformed into E. coli. Qualitative and quantitative growth assays utilizing these strains, along with previously engineered strains harbouring hicAB and mazEF constructs, revealed a lack of FitB or MazF-mediated toxicity in E. coli. In contrast, a HicA-mediated growth arrest effect, which could be abolished by co-expression of HicB, was detected, thus providing confirmation that the N. gonorrhoeae FA1090 genes, NGO1627/1628, encode a functional HicAB TA system. Subsequently, the HicA residues His24, His29, or His40, respectively, were changed to alanine residues by site-directed mutagenesis. Quantitative growth analysis confirmed that the His24 and His29 residues, but not His40 of gonococcal HicA are required for toxicity. Mutagenesis of the three TA systems was undertaken where both toxin and antitoxin genes were deleted and replaced by a kanamycin cassette in N. gonorrhoeae FA1090. Furthermore, complemented derivatives of FA1090ΔhicAB that expressed IPTG-inducible HicA, HicB or HicAB, respectively, were also generated. The growth characteristics of the complemented strains (FA1090ΔhicAB:hicA, FA1090ΔhicAB:hicB and FA1090ΔhicAB:hicAB) were examined in comparison to the wild-type and mutant strain. The results confirmed that all strains grew as the wild-type, either with induction with IPTG or not, with the exception of FA1090ΔhicAB:hicA induced with IPTG which exhibited growth arrest as judged by OD measurements. Another study finding was confirmation of hicAB gene expression in wild-type FA1090 during in vitro growth by extraction of total RNA and reverse transcription polymerase chain reaction (RT-PCR) analysis to detect specific mRNA transcripts. The use of specific hicA and hicB primers confirmed expression of both genes, and the combination of a hicA forward and hicB reverse primer provided evidence that both genes are co-transcribed. A final finding came when the total RNA preps were examined using an Agilent Bioanalyser. Notably, a doublet 16S rRNA peak was apparent in total RNA prepared from FA1090ΔhicAB:hicA strain induced with IPTG, but not the uninduced strain, suggesting interaction (or cleavage) of 16S rRNA by HicA, and potentially giving an insight into the mechanism by which gonococcal HicA influences bacterial growth and viability.19 0Item Restricted Evaluating the Efficacy of Laser-Assisted Endodontic Treatment: Insights from In Vitro Models and In Vivo Studies(University of Pennsylvania, 2024-08) Mominkhan, Dana; Teles, FlaviaObjective: To develop a clinically relevant complex multi-species biofilm model of endodontic infection and evaluate the performance of new laser technologies in vitro and in vivo. Methods: For the in vitro studies, bacterial species were selected based on the literature and the correlation network analysis (CNA) of our collection of 206 root canal samples. Biofilms were developed for 7 and 14 days. Three tooth models (SM, DMK, SMS) were compared using 200 extracted human premolars and four different laser technologies were tested. For the in vivo studies, patients presenting apical periodontitis were selected for treatment using the standard of care (NaOCl, control) or adjunctive laser treatment (test). Outcomes measured included CFU reduction and pain assessment. Results were evaluated using culture, SEM, FISH and CLSM LIVE/DEAD analysis. Data were analyzed using Kruskal-Wallis, PCA and GEE. Results: Tooth models were assessed with E. faecalis biofilms and tested using a multi-species biofilm composed of A. viscosus, F. nucleatum, P. micra, P. nigrescens and S. sanguinis. DMK was the most effective tooth model. EdgePRO™ was the most effective laser in the presence of NaOCl and LEAP™ in its absence. Fifty-two patients completed the study. CFUs were reduced by both treatments. No differences were observed regarding post-operative pain. Conclusion: The new multi-species biofilm mode developed here is a clinically relevant and effective tool to assess new treatments. EdgePRO™ is effective in reducing CFUs of in vitro and in vivo biofilms.28 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 0