Comprehensive analysis of antibiotic susceptibility across Variovorax to prepare for genetic analysis of horizontal gene transfer between strains.

Genetic manipulation of bacteria often involves the use of recombinant DNA techniques such as heterologous gene expression from plasmids and insertional mutagenesis. In order to effectively use these techniques it is necessary to use selectable markers, the most common of these being antibiotics. In our lab we commonly use two antibiotics, Kanamycin and Gentamicin, to select for recombinant strains. As we expand our studies other isolates within the genus Variovorax, it is necessary to text for susceptibility to these compounds to ensure that we can use them for selection. In this work we report the results of minimum inhibitory concentration (MIC) experiments using these two antibiotics against multiple Variovorax isolates in a broth microdilution assay. The strain was first inoculated into YE broth and incubated for about 24 hours prior to MIC testing. The diluted antibiotics, Kanamycin and Gentamicin, were prepared in 96-well plates with concentrations spanning from 1024 µg/mL- 0.5 µg/mL. Rows A-D were treated with Kanamycin, while rows E-H were treated with Gentamicin. After performing dilution across all columns, all wells were inoculated with a standardized bacterial dose. Column 12 was left free of antibiotic and acted as a positive control for bacterial growth, and rows D and H were uninoculated to ensure the test medium was sterile. Incubation took place at 30°C for 24–48 hours without shaking, and the endpoint growth was observed by eye and measured using a Spectramax250 plate reader. The MIC determined to be the lowest concentration of antibiotic that completely inhibited visible growth. This experiment will be the basis going forward for subsequent genetic experiments using these strains.