For a culture-based approach to surveillance, students start with the Core PARE Module in which soil is tested for the prevalence of live bacteria that are resistant to tetracycline and upload results to our database. From there, students in biosafety level 2-equipped laboratories can sub-culture resistant colonies and do further tests.
16S PCR from colony
Students choose a colony isolated from the core PARE module for tentative phylogenetic characterization. Genomic DNA is isolated and used in PCR reactions to amplify the 16S rRNA gene. Amplification results are confirmed by gel electrophoresis, the amplified DNA cleaned and sent for sequencing. Sequences are compared to those in the Ribosomal Database Project database to assign putative identity. Identity information is a critical early step to identify potential pathogens so this module is recommended prior to any future modules that require tetracycline-resistant colony sub-culturing.
This module was developed in collaboration with Dr. Brad Goodner, Hiram College.
Kirby-Bauer testing TcR isolates
Students assess colonies isolated in the core PARE module for resistance to other antibiotics using the Kirby-Bauer disk diffusion assay which relies on measurement of the zone of inhibition after microbial growth around an antibiotic impregnated filter disk. Since the Kirby-Bauer test is intended for use on isolates of known identity, it is strongly suggested that students perform 16S tentative identification on test isolates prior to this module.
This module was developed in collaboration with Dr. Adam Kleinschmit, Dubuque University.
Isolation of TcR plasmid and transfer into E. coli
Students learn about the genetics of plasmid transfer by isolating plasmids from a tetracycline-resistant (TcR) colony identified in the Core PARE module and then attempting to transform a tetracyline-sensitive strain of E. coli using the plasmid preparation. There is no assurance that TcR is harbored on a plasmid or that the transformation will be successful, but this module provides a good opportunity to discuss horizontal transfer in bacteria and its role in transmission of antibiotic resistance.
This module was developed in collaboration with Dr. Jennifer Larson, Capital University.