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.