The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA₂B₂ efflux pump-mediated macrolide resistance

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA₂B₂, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA₂B₂ in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA₂B₂ pump was completely abrogated by tonB123 deletion. In addition, macA₂B₂ expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA₂B₂-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA₂B₂-mediated macrolide efflux.