Rifampicin, an antibiotic that inhibits transcription by interacting with RNA polymerase (RNAP), has potent antistaphylococcal activity and is recommended as part of combination therapy for serious S. aureus infections and carriage eradication. Rifampicin resistance (RifR) mutations are located exclusively on the rpoB gene that encodes the β-subunit of RNAP. In addition to resistance, some RifRmutations can have pleiotropic effects by altering the RNAP transcription kinetics. We have uncovered broader negative consequences of RifR in S. aureus, where it showed that a RifR mutation, in clinical S. aureus caused persistent infection by both attenuating host innate immune responses and increasing cross resistance to other last-line of antibiotics. The aim of this work is to investigate the mechanisms and relationships between common clinical RifR mutations, persistent infection and co-resistance to other antimicrobials in S. aureus. In this study, 4177 S. aureus sequenced genomes were screened for mutated rpoB. A broad repertoire of different rpoB alleles was detected (n=180). By analyzing their prevalence and distributions among S. aureus lineages, we identified a sub-set of highly prevalent mutations associated with a strong positive selection signature. By introducing these rpoBmutations to S. aureus, we found that the most prevalent mutation promoted the emergence of a small colony variant (SCV) sub-population, which is a recurrent phenotype among persistent clinical isolates. Surprisingly, we found that these SCV correlate with the strong activation of a prophage encoding genes that modulate host innate immune responses. By finding new associations between RifR, SCV and the activation of prophage implicated in immune modulation this work is revealing a new mechanism by which S. aureus antibiotic resistance can promote persistent infection.