The Department of Biology witnessed the public defense of a PhD dissertation in Pure Sciences by researcher Mohammed Hassan Abdul-Kadhim Al-Kaabi, entitled: “Effect of Silver Nanoparticles on Gene Expression of Quorum Sensing Genes in Escherichia coli”
The dissertation was supervised by Dr. Suad Khalil Ibrahim and evaluated by a scientific committee comprising Dr. Saba Talib Hashim, Chair; Dr. Huda Suhail Abd, Member; Dr. Issam Jasim Khuraibit, Member; Dr. Mohammed Mahdi Jawad, Member; Dr. Wafaa Sabri Mahood, Member; and Dr. Suad Khalil Ibrahim, Member and Supervisor.
The study aimed to investigate the effects of green-synthesized silver nanoparticles loaded with pine seed oil on the gene expression of quorum sensing-related genes and biofilm formation in Escherichia coli isolated from clinical cases. The research also sought to assess the potential of these nanoparticles as anti-virulence agents that could contribute to reducing bacterial communication and biofilm-associated pathogenicity.
The study involved the collection of 155 clinical samples, including 85 urine samples from individuals with urinary tract infections (UTIs) and 70 samples from patients with diarrhea. The samples were collected from Babylon Teaching Hospital, Al-Qasim General Hospital, and Al-Hashimiya General Hospital in Babylon Governorate between 20 January and 20 March 2024. Morphological, microscopic, and biochemical examinations were performed, followed by confirmatory identification using the VITEK-2 system, resulting in the identification of 75 E. coli isolates.
Antimicrobial susceptibility testing was conducted using the disk diffusion method against ten antibiotics. The findings revealed high levels of antimicrobial resistance among the investigated isolates, with resistance rates of 100% to Amoxicillin, 84% to Ampicillin, 40% to Piperacillin, 88% to Cefepime, 60% to Ceftriaxone, 4% to Imipenem, 92% to Ceftazidime, 96% to Nitrofurantoin, 88% to Colistin, and 84% to Trimethoprim. These findings highlight the importance of exploring innovative complementary strategies targeting bacterial virulence and biofilm-associated mechanisms.
The study also investigated the production of Homoserine L-lactone (HSL) associated with quorum sensing mechanisms in E. coli. Aspartic acid at a concentration of 1% was incorporated into the culture media, and HSL production was assessed at different incubation periods. The results indicated that the optimal accumulation of HSL occurred after 4 and 24 hours of incubation, with its production further confirmed by microscopic examination.
Regarding biofilm formation, the Microtiter Plate Method (MTP) demonstrated that all 75 E. coli isolates were capable of biofilm formation, representing a rate of 100%. The isolates exhibited different levels of biofilm-forming capacity: 33 isolates (44%) were strong biofilm producers, 33 isolates (44%) showed moderate biofilm formation, and 9 isolates (12%) demonstrated weak biofilm-forming capacity.
At the molecular level, 25 bacterial isolates were selected for molecular analysis to detect genes associated with quorum sensing and biofilm formation. The LuxS gene was detected in 100% of the isolates, whereas the lsrR gene was detected in 64%. Meanwhile, both HslT and soxS, which were investigated in relation to biofilm formation, were detected in 96% of the examined E. coli isolates.
As part of the nanotechnology component of the research, silver nanoparticles loaded with pine seed oil were prepared using a green synthesis approach and characterized using several advanced techniques. Scanning electron microscopy (SEM) revealed predominantly spherical nanoparticles with sizes ranging approximately from 48.20 to 69.54 nm. X-ray diffraction (XRD) analysis showed characteristic diffraction peaks confirming the crystalline structure of the synthesized silver nanoparticles. Energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of silver, oxygen, and carbon in the analyzed sample. Atomic force microscopy (AFM) further demonstrated the spherical surface morphology of the nanoparticles and their relatively good distribution within the medium.
To investigate the molecular effects of the nanoparticles, sub-minimum inhibitory concentrations (sub-MICs) of pine seed oil (1 mg/mL) and silver nanoparticles (32 µg/mL) were used. Gene expression was quantified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR), with the 16S rRNA gene used as a reference gene.
The findings demonstrated a statistically significant reduction in the expression levels of LuxS, lsrR, HslT, and soxS in isolates treated with the silver nanoparticles, as well as in isolates treated with a combination of the nanoparticles and the antibiotic Amoxicillin (AMX), compared with treatment using pine seed oil alone. The observed differences were statistically significant at p≤0.05.
The findings indicate that green-synthesized silver nanoparticles exhibited greater efficacy in suppressing the expression of quorum sensing and biofilm-associated genes, particularly when combined with Amoxicillin, compared with pine seed oil alone. These results highlight the promising potential of green nanotechnology for developing anti-virulence strategies that target bacterial communication and biofilm formation rather than relying solely on inhibition of bacterial growth.
The study recommended conducting broader investigations to evaluate the efficacy of pine seed oil-loaded silver nanoparticles against other clinically important bacterial species. It also recommended examining different nanoparticle concentrations and doses to determine optimal efficacy while minimizing potential cytotoxicity to human cells. Comprehensive in vivo toxicity studies were recommended to establish the safety of the green-synthesized nanoparticles before their consideration for medical applications.
Further recommendations included investigating the synergistic effects between silver nanoparticles and different antibiotics, as well as examining their effects on quorum sensing proteins and cellular signaling pathways, rather than focusing exclusively on gene expression. The study also proposed evaluating the effects of the nanoparticles at different stages of biofilm development through advanced imaging techniques and molecular simulation approaches.
The dissertation further emphasized the importance of promoting green synthesis techniques for nanoparticle production because of their potential for safer and more sustainable production compared with conventional chemical synthesis methods. It also highlighted the future possibility of developing pharmaceutical formulations based on silver nanoparticles loaded with natural plant oils as anti-virulence agents for the management of bacterial infections, subject to further biological and toxicity investigations.
The research contributes to several United Nations Sustainable Development Goals (SDGs), most notably:
SDG 3 – Good Health and Well-Being: By exploring innovative approaches to reduce bacterial virulence and biofilm formation and by contributing to scientific efforts addressing antimicrobial resistance.
SDG 9 – Industry, Innovation and Infrastructure: Through the application of nanotechnology, molecular techniques, and innovative biomedical research approaches.
SDG 12 – Responsible Consumption and Production: Through the adoption of green synthesis as a more sustainable approach to nanoparticle preparation compared with conventional chemical methods.
SDG 6 – Clean Water and Sanitation: Indirectly, through advancing scientific knowledge related to clinically important microorganisms and strategies for controlling biofilm-forming bacteria.
SDG 17 – Partnerships for the Goals: Through the integration of microbiology, nanotechnology, molecular biology, and biomedical research in addressing complex health challenges.
This doctoral research represents an interdisciplinary approach combining molecular microbiology, nanotechnology, and green synthesis, demonstrating the potential of advanced scientific research to address contemporary health challenges and providing a foundation for future studies aimed at developing complementary therapeutic strategies against pathogenic and biofilm-forming bacteria.
