Utilizing single culture growth curves of Klebsiella pneumoniae isolates for fosfomycin dose-dependent gene expression
Utilizing single culture growth curves of Klebsiella pneumoniae isolates for fosfomycin dose-dependent gene expression Jessica F. Boccio, Morgan L. Bixby, Lindsey Collins, Ellora Daley, Elizabeth B. Hirsch University of Minnesota College of Pharmacy, Minneapolis, MN Background: Fosfomycin is an antibiotic used to treat urinary tract infections caused by Escherichia coli; however, it has not been approved for other Enterobacterales, such as K. pneumoniae, with fosfomycin resistance mechanisms such as fosA. To study the expression of fosA, we conducted growth curves for K. pneumoniae isolates to identify their logarithmic phases of growth and optimal fosfomycin exposure for RNA extraction. Materials/Methods: A starting inoculum of 105 CFU/mL in 25 mL of Mueller-Hinton broth (MHB) using overnight subcultures of three K. pneumoniae isolates (209, 254, 275) was prepared. Flasks were placed in a 37° water shaker bath and samples were taken in duplicate at hours 0, 1, 2, 3, 4, 6, 8, and 24, diluted, and plated to determine CFU/mL at each time point. Time points were graphed to identify the logarithmic phase of growth, which was used to determine the incubation time to inoculate MHB supplemented with three different concentrations of fosfomycin (0 µg/mL, 8 µg/mL, and 350 µg/mL). RNA was stabilized and extracted from these isolates. Results: All K. pneumoniae isolates’ logarithmic growth phases began at ~2 hours and ended at 6-8 hours. All RNA extracted from isolate 209, and from 254 and 275 at 8 µg/mL was within an acceptable quality range (+/-0.15 of the A260/280 value 2.00). Other extractions were not. Conclusion: Moving forward, extracted RNA will be used to perform reverse transcription quantitative polymerase chain reaction (RT qPCR) to examine the expression of fosA at various fosfomycin concentrations during growth.