Supplementary Material for: Increased Quinolone-Resistant Mutations of gyrA and parC Genes after Pouchitis Treatment with Ciprofloxacin

<b><i>Background:</i></b> Oral antibiotics, such as ciprofloxacin (CFX), are widely used for the treatment of acute and chronic pouchitis. Most bacterial mutations that confer quinolone resistance are at Ser-83 and Asp-87 in the <i>gyrA</i> gene and Ser-80 and Glu-84 in the <i>parC</i> gene. <b><i>Methods:</i></b> We obtained 51 stool samples from 43 patients who were diagnosed with ulcerative colitis and underwent ileal pouch-anal anastomosis. Patients were divided into 2 groups: 13 patients with CFX treatment of pouchitis and 30 patients without pouchitis. After extraction of fecal DNA, the amount of <i>Escherichia coli 16S rRNA</i>, <i>gyrA</i>, and <i>parC</i> gene DNA were measured using real-time polymerase chain reaction (PCR). Possible mutations at <i>gyrA</i> 83 and 87 and at <i>parC</i> 80 and 84 were investigated by PCR cloning and sequencing, and mutation rates were quantified by rapid PCR-restriction fragment length polymorphism. <b><i>Results:</i></b> Samples from both CFX-treated and -untreated patients had comparable levels of <i>gyrA</i> and <i>parC</i> gene DNA. Nucleic acid and amino acid mutations were identified at <i>gyrA</i> 83 and 87, and at <i>parC</i> 80 and 84. We successfully quantified mutation rates at <i>gyrA</i> 83 and 87, and at <i>parC</i> 84, all of which were significantly higher in samples from CFX-treated patients (70, 84, and 38%) than from CFX-untreated patients (13, 11, and 5%). <b><i>Conclusion:</i></b> <i>E. coli</i> in patient pouches may have mutations in their <i>gyrA</i> and <i>parC</i> genes that produce CFX resistance. Mutation rates of these genes were significantly higher in samples from CFX-treated patients. This study contributes to understanding the decrease and loss of CFX effectiveness against pouchitis.