Efflux pumps in Chromobacterium species and their involvement in antibiotic tolerance and survival in a co-culture competition model

ABSTRACT Members of the Chromobacterium genus includes opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis. Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis. We also demonstrate that adding sublethal bactobolin concentrations to the co-culture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show the CseAB-OprN genes are transcriptionally induced in cells exposed to sublethal bactobolin concentrations and that this causes an increase in bactobolin tolerance. Induction of this pump is through a bactobolin-responsive regulator, CseR. We also demonstrate that CseAB-OprN is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of specificities. We examine the distribution of CseAB-OprN and CdeAB-OprM in members of the Chromobacterium genus and find that CseAB-OprN is limited to the non-pathogenic C. subtsugae strains, whereas CdeAB-OprM is more widely distributed among members of the Chromobacterium genus including the occasional pathogen C. violaceum. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and use co-culture competition experiments to highlight the importance of efflux pumps in saprophytic bacteria. IMPORTANCE Antibiotic efflux pumps are best known for their role in increasing antibiotic resistance of a variety of pathogens. However, the role of these pumps in saprophytic species is much less well defined. This study describes the identification and characterization of two predicted efflux pump gene clusters in members of the Chromobacterium genus, which is primarily comprised of non-pathogenic saprophytes but also has several members known to occasionally cause highly fatal infections in humans. One of the predicted efflux pump gene clusters is present in every member of the Chromobacterium genus and shown to increase resistance to a broad range of antibiotics, including those that are clinically relevant. The other gene cluster confers resistance to a limited range of antibiotics and is found only in Chromobacterium subtsugae, a subset of this genus that is entirely nonpathogenic. We demonstrate these pumps can be activated by their antibiotic substrates to increase antibiotic tolerance. We also use a dual-species laboratory model to demonstrate that efflux pump activation by antibiotics is important for C. subtsugae to survive during competition with another antibiotic-producing bacteria. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside of the host.