Shigella sonnei evolution experiment

To examine how continuous exposure to (fluoro)quinolone may impact on the evolution of Shigella sonnei, we selected a representative isolate from each of the fluoroquinolone resistant (CenAsiaIII) and the susceptible (GlobalIII) clades. Both isolates, cipR-VN 02-1157 (gyrA-S83L, parC-S80I, and gyrA-D87G) and cipS-VN 03-0142 (gyrA-S83L), were isolated in 2015 as part of a hospital-based diarrheal surveillance study conducted in Ho Chi Minh City, Vietnam. Isolates were selected based on sequencing quality and the absence of plasmids encoding resistance to 3rd generation cephalosporins and macrolides (to discount potential interactions). M9 minimal was used as culture medium for the experiment to limit over-proliferation. CipR-VN and cipS-VN were subjected to three longitudinal culture conditions in duplicate: i) M9 (glucose + niacin) medium only, ii) M9 (glucose + niacin) supplemented with 256 µg/mL of nalidixic acid, and iii) M9/B3 (glucose + niacin) supplemented with half the appropriate MIC of ciprofloxacin (0.125 µg/mL for cipS-VN and 4 µg/mL for cipR-VN). Cultures were incubated at 37oC with continuous agitation for eight weeks; a sub-culture was performed every 24 hours by transferring 20µL into 5mL of fresh medium. This process resulted in approximately eight bacterial generations (log2[5,000/20]) per 24 hours. At the end of the experiment (D56), organisms were harvested from 2mL culture from each of the twelve experiments. Total genomic DNA was extracted from each sample using the Wizard Genomic DNA Extraction kit (Promega), and these were predicted to represent S. sonnei population diversity within each condition.