Chronic Exposure to Low Concentration of Graphene Oxide Increases Bacterial Pathogenicity via the Envelope Stress Response

Graphene oxide (GO), which has diverse antimicrobial mechanisms, is a promising material to address antibiotic resistance. Considering the emergence of antibiotic tolerance/resistance due to prolonged exposure to sublethal antibiotics, it is imperative to assess the microbiological effects and related adaptive mechanisms under chronic exposure to sublethal levels of GO, which have rarely been explored. After repetitive exposure to 5 mg/L GO for 200 subcultures (400 days), evolved Escherichia coli (E. coli) cells (E_GO) differed significantly from their ancestor cells according to transcriptomic and metabolomic analyses. Contact with GO surfaces transformed E. coli by activating the Cpx envelope stress response (ESR), resulting in more than twofold greater extracellular protease release and biofilm formation. The ESR also modulated the envelope structure and function via increases in membrane fluidity, permeation, and lipopolysaccharide content to fulfill growth requirements and combat envelope stress. As a consequence of metabolic adjustment, E_GO cells showed advantages of surviving in an acidic and oxidative environment, which resembles the cytosol of host cells. With these adaptive features, E_GO cells exhibited higher pathogenicity than ancestor E. coli cells as evidenced by increased bacterial invasion and intracellular survival and a more severe inflammatory response in macrophage cells. To conclude, we seek to raise awareness of the possible occurrence of microbial adaptation to antimicrobial nanomaterials, which may be implicated in cross-adaptation to harsh environments and eventually the prevalence of virulence.