Efflux pumps in Chromobacterium species and their involvement in antibiotic tolerance 2 and survival in a co-culture competition model

(250 words) Members of the Chromobacterium genus includes opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium 27 subtsugae (formerly C. violaceum ) strain CV017, we identified a resistance nodulation division 28 (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics including 29 the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis . Here, we 30 show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model 31 with B. thailandensis. We also demonstrate that adding sublethal bactobolin concentrations to the 32 co-culture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, 33 the increased survival requires a second, previously unreported pump we call CseAB-OprN. We 34 show the CseAB-OprN genes are transcriptionally induced in cells exposed to sublethal 35 bactobolin concentrations and that this causes an increase in bactobolin tolerance. Induction of 36 this pump is through a bactobolin-responsive regulator, CseR. We also demonstrate that CseAB- 37 OprN is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader 38 range of specificities. We examine the distribution of CseAB-OprN and CdeAB-OprM in members 39 of the Chromobacterium genus and find that CseAB-OprN is limited to the non-pathogenic C. 40 subtsugae strains, whereas CdeAB-OprM is more widely distributed among members of the 41 Chromobacterium genus including the occasional pathogen C. violaceum. Our results provide 42 new information on the antibiotic resistance mechanisms of Chromobacterium species and use 43 co-culture competition experiments to highlight the importance of efflux pumps in saprophytic 44 bacteria. 45 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 65 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 the low doses of bactobolin. However, increased survival is not dependent on CdeAB-OprM. Instead, we find a newly identified pump, CseAB-OprN, and show this pump is important for the increased survival in co-culture. We also demonstrate the involvement of CseAB- OprN in a specific response to bactobolin and that this gene cluster is activated by sublethal bactobolin concentrations through a LysR-family regulator encoded adjacent to the cseAB-oprN genes, CseR. We show that the cseAB-oprN genes are limited to the C. subtsugae strains, which are not known to be pathogenic, and that the genes encoding CdeAB-OprM are widely distributed in the Chromobacterium genus. Together, our results describe two RND-efflux pumps found in 130 members of the Chromobacterium genus and demonstrate how the evolution of antibiotic response might be important during competition with other species in the soil. demonstrate antibiotic specificity and regulation of two efflux pumps of subtsugae CV017. One of these, CseAB-OprN, not previously is limited the C. subtsugae species. The other, CdeAB-OprM, is widely distributed members of the Chromobacterium genus. CdeAB-OprM has a broad range of specificity for such as tetracycline, erythromycin, chloramphenicol and ciprofloxacin. As ciprofloxacin of the recommended treatments for infections with pathogenic Chromobacterium suggest CdeAB-OprM might be important in cases of antibiotic failures during infections. To date, there are very few on Chromobacterium mechanisms of antibiotic resistance and drug export Results of this study will be useful for identifying resistance mechanisms in clinical isolates and for future Chromobacterium research. As Chromobacterium members are primarily saprophytic non-pathogens, our results are also important for understanding the ecology and evolution of efflux pumps. Our results demonstrate that both CdeAB-OprM and CseAB-OprN are induced by their antibiotic substrates. Such a response is typical of related pumps as