Unfortunately, it may be more difficult to predict susceptibility than resistance using RBFE, and for rpoB only one susceptible mutation could be confidently predicted. If we assume that most susceptible mutations will not affect the binding affinity for the antibiotic, then they would have a DDG of zero. The predicted DDG of such mutations would hence require the estimated error to be at least less than the value of the ECOFF (for rpoB and DNA gyrase 1.2 and 0.9 kcal/mol, respectively) to make a confident susceptible prediction. The magnitude of error for the mutations in this study was greater than the relevant ECOFF in all but one case (rpoB S388L, Table ). It is likely that the error could be reduced by running a greater number of repeats, however some mutations can result in a small increase in MIC but not enough to confer resistance (as susceptibility can be defined as any MIC up to the ECOFF), and in such cases even a lower level of error (± 0.5 kcal/mol) may still prove insufficient for prediction.