Escherichia coli (E. Coli), is a prominent hospital-acquired bacterial infection that has grown resistant to antibiotics partially due to their misuse and overprescription. To combat this public health issue, many studies have focused on understanding the antibiotic activity of naturally occurring compounds. Two such examples include carvacrol and thymol, natural compounds found in oregano and thyme, which have shown the ability to inhibit bacterial growth with some studies suggesting disruption of the cell membrane as a possible mechanism of action. However, it is still unknown if interactions with other cellular molecular targets occur. In this study, we aimed to develop a methodology for a course undergraduate research experience (CURE) module in an introductory biochemistry laboratory course where students test the antibacterial activity of natural compounds in herbs. Our goals were for students to investigate the in vitro antibacterial efficacy of natural compounds against E. coli, and if these compounds bind to the A-site ribosomal RNA (rRNA) as another mode of antibacterial mechanism like other antibiotics such as paromomycin. We tested carvacrol and thymol during the development of these protocols. The efficacy of the compounds was tested by performing a broth microdilution to determine the MIC (minimum inhibitory concentration) while binding to A-site rRNA was tested via fluorescence assay. The MIC experiments results show that carvacrol and thymol inhibit E. coli growth at similar concentrations. Preliminary fluorescence assay results showed that thymol binds to the A-site rRNA suggesting disruption of protein synthesis as another mode of action, facilitating bacterial cell death. These protocols were successfully optimized to be carried out once a week during the laboratory meetings. As part of the CURE module, students will be implementing these protocols as they test other natural compounds found in herbs while also developing laboratory skills such as handling RNA, making solutions, carrying out fluorescence assays, and data analysis.

Anette Casiano-Negroni

Trinity University

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