Pseudo-resistant Bacillus cereus uses biofilm-related mechanism to mimic vancomycin resistance during agar diffusion susceptibility testing

The glycopeptide vancomycin is a drug of choice for the treatment of severe non-gastrointestinal infections with members of Bacillus cereus sensu lato. Recently, sporadic detection of vancomycin resistant phenotypes emerged, mostly for agar diffusion testing. The food packaging isolate BC70 displayed a pseudo-resistant phenotype for vancomycin in both Etest and disk diffusion. In this work, we used RNA-Seq on the nanopore platform to study differentially expressed genes in BC70 cells, which were able to actively move into the inhibition zone during vancomycin susceptibility testing using Etest and therefore appeared to be resistant. Transcriptomic analysis revealed several key genes of biofilm formation (e.g. calY, tasA, krsEABC) to be upregulated in pseudo-resistant cells suggesting a biofilm-related mechanism of motility. Downregulation of virulence (e.g. hblABCD, nheABC, plcR) and flagellar genes compared to swarming cells also confirmed the non-swarming phenotype of the pseudo-resistant isolate. This type of biofilm-related motility was distinct from swarming motility and joined the list of bacterial strategies interfering with reliable antimicrobial susceptibility testing. We compared the gene expression of pseudo-resistant BC70 with the reference strain ATCC 14579, which did not show resistance during vancomycin susceptibility testing. Furthermore, gene expression of BC70 was compared to swarming BC70 cells as well as BC70 cells on MH agar.