Non-destructive monitoring of antibiotic action in Bacillus subtilis biofilms using dual-parameter swept-source optical coherence tomography

Biofilms show an enhanced tolerance to antimicrobials compared to planktonic cells and conventional susceptibility tests provide limited insight into antibiotic action within intact biofilms. We assessed swept-source optical coherence tomography (SSOCT) as a non-destructive, label-free platform for biofilm monitoring, extracting two label-free parameters, the optical attenuation coefficient (μt) and biospeckle temporal contrast (γ). Biofilms cultured for 24 h, were treated with antibiotics at minimum biofilm eradication concentration (MBEC) determined by the Calgary biofilm device and imaged at 0, 24 and 48 h post-treatment alongside time-matched untreated controls, with parallel colony forming unit (CFU) enumeration. Untreated biofilms showed progressive increases in both μt and γ, confirming that time-matched rather than pre-treatment controls are required. Treated biofilms showed elevated μt (76.9-103.2% above control, p < 0.001) but declining γ (68.4% and 68.6% below control at 48 h, p < 0.001), with the μt-γ correlation reversing from positive during maturation (r = +0.93) to negative under treatment (r = −0.94). Viable counts decrease by ∼2.5 log10 CFU/mL in both treated arms (p < 0.001), confirming substantial killing; γ nonetheless declined nonlinearly with respect to viability and continued falling after CFU/mL stabilized, indicating that γ reflects progressive physical quiescence rather than viable cell numbers. SSOCT attenuation and biospeckle thus offer a non-destructive complement to conventional viability assays.