Chloramphenicol-mobilized Bacillus subtilis transiently expresses resistance to multiple antibiotics, including the glycopeptides phleomycin and bleomycin

ABSTRACT Antibiotic resistance presents an urgent global crisis, exacerbated by antibiotic overuse. Understanding the regulation of resistance genes within bacterial populations can inform strategies to prevent the spread of antibiotic resistance, and reveal how antibiotics shape microbial communities. We identified upregulation of five antibiotic resistance loci in Bacillus subtilis colonies on solid growth media, following exposure to subinhibitory chloramphenicol concentrations. Notably, four resistance loci, bmrCD, vmlR, tlrB, and ytbDE, are regulated by transcription attenuation. Full expression depends upon antibiotic-induced ribosome stalling on upstream leader peptides, promoting transcription of the downstream gene. Here, we use luciferase reporter constructs fused to the 5′ regulatory region of each resistance gene to show differential spatiotemporal patterns of antibiotic resistance gene expression, revealing an intrinsic activation in addition to chloramphenicol induction in mobilized B. subtilis colonies. Because expression is under translational regulation, the data suggest natural translation pausing, in addition to antibiotic exposure, is an endogenous function that regulates these antibiotic resistance genes. While VmlR and TlrB have been previously characterized as conferring resistance to LS A Ps (lincosamides, streptogramin A, and pleuromutilin) and tylosin, respectively, antibiotics for BmrCD and YtbDE resistance complexes have not yet been identified. We demonstrate that although these resistance genes do not provide resistance to chloramphenicol, pre-exposure to chloramphenicol improved B. subtilis growth when cells were subsequently exposed to subinhibitory concentrations of respective antibiotics. We discovered that BmrCD confers resistance to the DNA-damaging glycopeptides, phleomycin and bleomycin, revealing that resistance arising from stalled ribosomes extends beyond drugs that target translation. IMPORTANCE Antibiotics are a natural component of bacterial communities that are vital to modern medicine and combating antibiotic-resistant pathogens. Understanding the roles antibiotics play in microbial populations provides insights into the origins of antibiotic resistance. Many studies show that subinhibitory concentrations of antibiotics stimulate bacterial gene expression, including antibiotic resistance, and influence community dynamics. This study shows that bacteria on an agar surface transiently elevate expression of intrinsic resistance genes for multiple antibiotics. Subinhibitory chloramphenicol exposure stimulates the bacterial population both to expand across the surface and to enhance expression of the resistance genes in a heterogeneous pattern. The results illustrate how exposure to some antibiotics substantially changes bacterial population dynamics that include the spatiotemporally controlled expression of endogenous antibiotic resistance.