'Supermutators' found amongst highly levofloxacin-resistant E. coli isolates: a rapid protocol for the detection of mutation sites.

Fluoroquinolone resistance is gradually acquired through several mechanisms. In particular, chromosomal mutations in the genes encoding topoisomerases II and IV and increased expression of the multidrug efflux pump AcrAB-TolC are the most common mechanisms. In this study, multiplex polymerase chain reaction (PCR) protocols were designed for high-throughput sequencing of the quinolone resistance determining regions of topoisomerases genes (gyrA, parC and parE) and/or the expression regulation systems of multidrug efflux pump AcrAB (acrRAB, marRAB and soxSR). These protocols were applied to sequence samples from five subpopulations of 103 clinical Escherichia coli isolates. These subpopulations were classified according to their levofloxacin susceptibility pattern as follows: highly resistant (HR), resistant (R), intermediate (I), reduced susceptibility (RS) and susceptible (S). All HR isolates had mutations in the six genes surveyed, with two 'supermutator' isolates harboring 13 mutations in these six genes. Strong associations were observed between mutations in acrR and HR isolates, parE and R/HR isolates and parC and I/R/HR isolates, whereas surprisingly, gyrA mutations were common in RS/I/R/HR isolates. Further investigation revealed that strong associations were limited to the triple mutations gyrA-S83L/D87N/R237H and HR isolates and the double mutations S83L/D87N and I/R/HR isolates, whereas the single mutation S83L was common in RS/I/R/HR isolates. Interestingly, two novel mutations (gyrA-R237H and acrR-V29G) were located and found to strongly associate with HR isolates. To the best of our knowledge, the gyrA-R237H and acrR-V29G mutations have never been reported and require further investigation to determine their exact role in resistance or 'fitness' as defined by their ability to compensate for the organismal cost of gaining resistance.