Isolation of novel Bacteriophages against *Streptococcus mutans*: A targeted approach to controlling Dental Caries

Description

Dental caries is one of the most prevalent biofilm-associated diseases worldwide and remains a major challenge in oral health. Among the microorganisms associated with dental caries, Streptococcus mutans is a particularly important cariogenic bacterium because of its ability to colonize tooth surfaces, metabolize dietary carbohydrates, produce extracellular polysaccharides, and establish persistent biofilms that promote the development of dental caries [1,2]. These characteristics make S. mutans an appropriate target for developing more selective approaches to controlling cariogenic bacterial populations. Bacteriophages are viruses that infect bacteria with a high degree of host specificity and therefore represent an attractive biological strategy for targeted bacterial control. In contrast to broad-spectrum antimicrobial approaches, phage-based strategies have the potential to selectively act against a defined bacterial target while reducing unnecessary disruption of other members of the microbial community. Previous studies have demonstrated that naturally occurring bacteriophages capable of infecting S. mutans can be recovered from biological samples and can exert measurable antibacterial and antibiofilm effects [1,2]. However, the number of characterized S. mutans bacteriophages remains relatively limited, creating an opportunity to explore additional phage diversity and identify isolates with distinct biological properties. This project will investigate the discovery, isolation, purification, and characterization of bacteriophages capable of infecting S. mutans from locally available environmental sources. A central objective is not simply to determine whether phages can be recovered, but to establish a systematic screening and characterization pipeline capable of identifying isolates with reproducible lytic activity and potential antibiofilm effects. The project will therefore generate experimentally characterized phage candidates rather than relying on a predetermined expectation of successful isolation. The research builds directly on the supervisor's established experience in bacteriophage research. Previous work by Ishnaiwer and Al-Razem demonstrated the recovery and characterization of bacteriophages from Laban Jameed, providing experience in phage discovery from complex biological and food-associated materials [3]. Subsequent research included the isolation and characterization of a bacteriophage targeting avian-pathogenic Escherichia coli, incorporating plaque-based detection, phage quantification, host susceptibility testing, and characterization of phage growth properties [4]. Additional work involved the isolation and characterization of a Salmonella Gallinarum bacteriophage [5]. The proposed study extends this research trajectory into oral microbiology by applying established bacteriophage isolation and characterization expertise to a clinically important cariogenic organism. The project will use a host-directed discovery strategy in which locally available environmental samples are screened for phage activity against S. mutans. Candidate phages will be enriched using susceptible bacterial hosts, detected through plaque-based assays, purified through repeated plaque isolation, and quantified using standard phage titration procedures. The resulting isolates will then undergo biological characterization, including assessment of lytic activity and susceptibility across available S. mutans strains. This approach will allow the students to distinguish individual phage isolates and identify candidates with reproducible and potentially broader activity. A major strength of the proposed work is the inclusion of biofilm-associated activity as a key characterization endpoint. Because the cariogenic potential of S. mutans is closely linked to its capacity to establish structured biofilms, demonstrating activity against planktonic bacteria alone would provide only a limited assessment of potential biological relevance. Selected phage isolates will therefore be evaluated against S. mutans biofilms using quantitative assays designed to determine whether phage exposure reduces biofilm formation and/or affects established biofilms. Previous research has demonstrated that S. mutans bacteriophages can reduce viable bacteria and metabolic activity within biofilms, providing a strong scientific rationale for incorporating this component into the proposed investigation [1]. The project is deliberately structured as an undergraduate research project rather than a routine laboratory exercise. Participating students will be involved in the complete research cycle, including formulation of experimental questions, preparation of bacterial cultures, environmental sample processing, phage enrichment, plaque detection, purification, titration, host-range assessment, biofilm experimentation, quantitative data collection, statistical interpretation, and scientific communication. Each student will undertake defined experimental responsibilities under faculty supervision while contributing to a common research objective. This structure will provide students with direct experience of how microbiological research progresses from an initial biological question through discovery, experimental validation, data analysis, and scientific dissemination. The expected primary outcome is a well-documented collection of newly isolated S. mutans-infecting bacteriophages, where successful isolation is achieved, accompanied by a structured dataset describing their infectivity, host range, and antibiofilm properties. Isolates demonstrating consistent lytic activity and reproducible effects against S. mutans biofilms will be prioritized as candidate phages for subsequent investigation. Importantly, the project does not require every environmental sample to yield a phage; unsuccessful samples will also provide scientifically interpretable information regarding phage recovery and host susceptibility. This discovery-oriented design therefore provides meaningful research outcomes across different experimental results. The project is also intended to establish a foundation for continued research beyond the PalUROP period. If promising phage isolates are obtained and sufficient biological characterization is achieved, selected candidates can subsequently undergo molecular or genomic characterization to investigate their taxonomic identity, genetic diversity, and potential determinants of host specificity and lytic activity. Such downstream work would extend the undergraduate project into a broader research program focused on phage–S. mutans interactions and targeted approaches to controlling cariogenic biofilms. Finally, the project is designed to produce tangible scientific outputs. The generated dataset, together with sufficiently characterized phage isolates and reproducible biological findings, may provide the basis for a manuscript targeting a peer-reviewed journal in bacteriophage research, microbiology, oral microbiology, or antimicrobial research. Students will also have the opportunity to prepare a scientific poster or oral presentation for dissemination through PalUROP and Palestine Polytechnic University. In this way, the project combines original bacterial-phage discovery, clinically relevant oral microbiology, hands-on undergraduate research training, and the potential generation of a research resource for future investigations. Methodology 1. Selection and preparation of S. mutans host strains Available S. mutans strains will be maintained and cultured under appropriate laboratory conditions and used as bacterial hosts for phage discovery. Where multiple strains are available, they will be incorporated into the screening workflow to increase the likelihood of identifying phages with different host specificities. Bacterial controls will be included throughout the experimental procedures. 2. Collection and preparation of potential phage sources Locally accessible environmental and biological samples will be selected as potential reservoirs of bacteriophages. Samples will be processed using standardized procedures appropriate to their physical characteristics before host-directed enrichment. The selection of locally available sources provides an opportunity to investigate previously unexplored phage diversity while keeping the project experimentally feasible for undergraduate researchers. 3. Host-directed enrichment of bacteriophages Processed samples will be enriched in the presence of actively growing S. mutans cultures to favor the recovery of phages capable of infecting the target bacterium. Following enrichment, bacterial cells and particulate material will be removed using appropriate clarification and filtration procedures. The resulting preparations will be screened for phage activity. This workflow builds on the supervisor's previous experience with host-directed bacteriophage enrichment and recovery from complex samples [3–5]. 4. Detection and isolation of phage candidates Potential phage activity will be detected using plaque-based assays. Plaques consistent with bacteriophage-mediated lysis will be individually recovered and subjected to repeated purification to obtain reproducible phage isolates. Appropriate bacterial-only controls will be included to distinguish phage-mediated lysis from nonspecific inhibition. Plaque morphology and formation will also be documented as preliminary phenotypic characteristics. 5. Phage quantification Purified isolates will be quantified using plaque-forming-unit assays to determine phage titers. Standardized titration will allow comparison among isolates and provide the quantitative basis for subsequent biological experiments. Each confirmed isolate will receive a unique identifier and will be maintained as part of a documented phage collection. 6. Biological characterization and host-range assessment Purified phages will be evaluated for reproducibility of lytic activity and tested against available S. mutans strains. Host-range analysis will determine whether individual isolates exhibit narrow strain specificity or activity across multiple S. mutans strains. Selected isolates will be compared according to plaque phenotype, infectivity, and consistency of bacterial lysis. 7. Evaluation of activity against S. mutans biofilms Phage isolates showing reproducible lytic activity will be investigated in S. mutans biofilm models. Experiments will assess their effects on biofilm formation and, where feasible, established biofilms. Quantitative measurements will be used to compare phage-treated and untreated controls. Appropriate experimental replication will be incorporated to determine whether observed effects are reproducible. 8. Data integration and selection of candidate phages Results from isolation, titration, host-range testing, and biofilm experiments will be integrated into a comparative dataset. Candidate phages will be selected according to predefined characteristics, including reproducible lytic activity, infectivity across tested S. mutans strains, and measurable antibiofilm activity. This step will identify isolates with the greatest potential for subsequent research rather than selecting candidates solely on the basis of plaque formation. 9. Reproducibility and quality control Key experiments will be performed with appropriate controls and replication. Experimental conditions, sample identities, phage isolates, titers, host susceptibility results, and biofilm measurements will be systematically recorded. Findings from promising isolates will be independently repeated before being considered reproducible. The supervisor will oversee experimental design, quality control, interpretation of results, and selection of isolates for subsequent characterization. 10. Potential downstream characterization If sufficiently promising isolates are obtained and the necessary institutional resources are available, selected phages may undergo additional molecular or genomic characterization in subsequent work. This may provide information on phage identity, genetic diversity, host specificity, and potential mechanisms underlying their activity. These analyses will constitute a potential extension of the project rather than a prerequisite for its successful completion. This methodology provides a clear progression from local phage discovery to experimentally validated candidate selection, while remaining achievable within an undergraduate research framework. It also builds on the supervisor's demonstrated experience in bacteriophage isolation and characterization [3–5] while introducing a new clinically relevant target and a stronger functional endpoint through evaluation of activity against S. mutans biofilms. References [1] Dalmasso M, de Haas E, Neve H, et al. Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms. PLoS ONE. 2015;10(9):e0138651. [2] Ben-Zaken H, Kraitman R, Coppenhagen-Glazer S, et al. Isolation and Characterization of Streptococcus mutans Phage as a Possible Treatment Agent for Caries. Viruses. 2021;13(5):825. [3] Ishnaiwer MM, Al-Razem F. Isolation and Characterization of Bacteriophages from Laban Jameed. Food and Nutrition Sciences. 2013;4:56–66. doi:10.4236/fns.2013.411A008. [4] Fakhouri H, Juneidi O, Al Mohtaseb H, Ishnaiwer M, Al-Razem F. Isolation and Characterization of a Bacteriophage That Hosts on Avian-pathogenic Escherichia coli (APEC). International Journal of Poultry Science. 2019;18(5):223–230. doi:10.3923/ijps.2019.223.230. [5] Al-Razem F, Al-Aloul H, Ishnaiwer M, Qadi R. Isolation and Partial Characterization of Salmonella Gallinarum Bacteriophage. Saudi Journal of Biological Sciences. 2022;29(5):3308–3312. doi:10.1016/j.sjbs.2022.02.007.