The following study is investigating the different GyrB mutations associated with Escherichia coli clinical isolates. The study interrogates part of the ATPase binding site (a.a 132-199) as it covers most of the naturally occurring mutations in GyrB. The following results were obtained: for Arg-136 two isolates had mutations, the first is isolate-1 (Ala-136), and the second is isolate-5 (Cys-136). Gly-164 had no changes for all tested isolates. For Thr-165 only isolate-3 had a change to Ser-165. Accuracy of sequence translation was checked by sequencing both CFT073 and MG1655. The current study presents novel mutations in the GyrB24 subdomain of the gyrase enzyme. These new mutations showed normal enzyme activity (no reduction in ATPase functions) indicating that they might be a result of GyrB interaction with ATP analog molecules rather than antibacterial agents such as coumarins. Furthermore, our findings are supporting the idea that mutations in the GyrB24 would require synchronization with the efflux pumps to maintain antibiotic resistance against coumarins. © 2018 Trans Tech Publications, Switzerland.