Development of a Biosafety-Certified Microbial Biofilm Inhibition Platform using Engineered Probiotics

Thesis Abstract

The increasing prevalence of healthcare-associated infections linked to resilient microbial biofilms on medical devices and chronic wound environments necessitates innovative, biosafety-rated strategies to prevent and disrupt biofilm formation without compromising host or environmental safety. This study investigates a biosafety-certified platform utilizing engineered probiotic strains to inhibit microbial biofilms through synergistic antagonism, quorum sensing disruption, and targeted anti-biofilm metabolite production, while complying with regulatory biosafety frameworks. The aim is to design, implement, and evaluate a scalable, biosafety-governed probiotic system capable of reducing biofilm biomass by at least 60% in clinically relevant settings and to identify operational parameters that optimize efficacy without eliciting adverse host responses. Specific objectives are (1) to engineer probiotic strains with controlled anti-biofilm activity, including CRISPR-based quorum sensing modulators and production of lactonase and bacteriocin cocktails; (2) to establish a biosafety-certified containment and delivery platform operative in simulated hospital environments; (3) to quantify biofilm inhibition on representative pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli) on medical-grade materials using standardized in vitro models; (4) to evaluate host-compatibility and environmental safety through in vitro cytotoxicity assays and microbiome impact assessment; and (5) to develop a decision-support framework for translational deployment. The methodological approach adopts an explanatory sequential mixed-methods design, combining empirical laboratory experiments with qualitative risk assessment and stakeholder perspectives. The population comprises engineered probiotic candidates (Lactobacillus rhamnosus GG and Lactobacillus plantarum WCFS1 derivatives) and clinically relevant biofilm-forming pathogens. A sample of 12 engineered strains will be generated and screened for stability, plasmid retention, and biosafety features. In vitro experiments will utilize standardized biofilm assays, including crystal violet biomass quantification, confocal laser scanning microscopy for structure analysis, and live/dead staining to assess viability. Biofilm models will include 96-well microtiter plates, CDC biofilm reactors, and silicone catheter segments to emulate device-associated biofilms. Data collection instruments include spectrophotometric readers for biomass (OD600/OD570), CLSM imaging, high-performance liquid chromatography (HPLC) for metabolite profiling, and quantitative PCR for gene expression of biofilm-related pathways. Safety assessments will employ cytotoxicity testing on human keratinocytes (HaCaT) and primary epithelial cells, along with analytical microbial risk assessment using 16S rRNA sequencing to monitor microbiome shifts. Data analysis will apply multivariate regression to correlate engineered strain traits with biofilm suppression, ANOVA to compare interventions across pathogens and materials, and pathway analysis for quorum-sensing and metabolite data. A conceptual framework will integrate the biosafety certification model (e.g., NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules) with anti-biofilm efficacy indicators to yield a translational readiness index. Ethical considerations address dual-use risk, biosafety compliance, and informed consent for any clinical collaboration. Expected findings include demonstrable reductions in biofilm biomass by ?60% across tested pathogens on multiple substrates, with substantial disruption of three-dimensional biofilm architecture as evidenced by CLSM. Engineered strains are anticipated to show stable performance with minimal horizontal gene transfer risk and no detectable cytotoxic effects on human cells. Metabolite profiling is expected to reveal a combination therapy of lactonase activity and bacteriocins contributing to synergistic inhibition. The study aims to contribute knowledge on how controlled probiotic engineering can provide a safe, deployable anti-biofilm strategy compatible with hospital infection-control practices, and to inform regulatory pathways for future clinical trials. The anticipated contribution to knowledge includes (i) a validated biosafety-certified platform for probiotic-mediated biofilm inhibition, (ii) empirical data on the efficacy of combined anti-biofilm modalities against major nosocomial pathogens, and (iii) a practical framework for evaluating translational readiness of engineered probiotics in device-associated infection prevention. The main conclusion will assert the feasibility of a biosafety-governed, engineered-probiotic approach as a complementary strategy to standard disinfection, with recommendations for scalable manufacturing, regulatory approval processes, and guidelines for integration into infection-control workflows.