Antimicrobial blue light (aBL) treatment is considered low risk for the development of bacterial resistance and tolerance due to its multitarget mode of action. The aim of the current study was to demonstrate whether tolerance development occurs in Gram-negative bacteria. We evaluated the potential of tolerance/resistance development in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa and demonstrated that representative Gram-negative bacteria may develop tolerance to aBL. The observed adaption was a stable feature. Assays involving E. coli K-12 tolC-, tolA-, umuD-, and recA-deficient mutants revealed some possible mechanisms for aBL tolerance development.

Enterococcus faecium and Enterococcus faecalis are opportunistic pathogens that can cause a vast variety of nosocomial infections. Moreover, E. faecium belongs to the group of ESKAPE microbes, which are the main cause of hospital-acquired infections and are especially difficult to treat because of their resistance to many antibiotics. Antimicrobial photodynamic inactivation (aPDI) represents an alternative to overcome multidrug resistance problems. This process requires the simultaneous presence of oxygen, visible light, and photosensitizing compounds. In this work, aPDI was used to resensitize Enterococcus spp. isolates to antibiotics. Antibiotic susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations was combined with synergy testing methods recommended by the American Society for Microbiology. Two clinical isolates, E. faecalis and E. faecium, were treated with a combination of aPDI utilizing rose bengal (RB) or fullerene (FL) derivative as photosensitizers, antimicrobial blue light (aBL), and 10 recommended antibiotics. aPDI appeared to significantly impact the survival rate of both isolates, while aBL had no significant effect. The synergy testing results differed between strains and utilized methods. Synergy was observed for RB aPDI in combination with gentamycin, ciprofloxacin and daptomycin against E. faecalis. For E. faecium, synergy was observed between RB aPDI and gentamycin or ciprofloxacin, while for RB aPDI with vancomycin or daptomycin, antagonism was observed. A combination of FL aPDI gives a synergistic effect against E. faecalis only with imipenem. Postantibiotic effect tests for E. faecium demonstrated that this isolate exposed to aPDI in combination with gentamycin, streptomycin, tigecycline, doxycycline, or daptomycin exhibits delayed growth in comparison to untreated bacteria. The results of synergy testing confirmed the effectiveness of aPDI in resensitization of the bacteria to antibiotics, which presents great potential in the treatment of infections caused by multidrug-resistant strains.

Antimicrobial blue light (aBL) has become a promising non-invasive method that uses visible light, typically within the 405–470 nm wavelength range, to efficiently inactivate a wide variety of pathogens. However, the mechanism of antimicrobial blue light (aBL) has not been fully understood. In this study, our research group investigated the sensitivity of Escherichia coli BW25113 single-gene deletion mutants to individual stressors generated by aBL. Sixty-four aBL-sensitive mutants were tested under conditions mimicking the stress generated by irradiation with aBL, with their growth defects compared to the wild-type strain. Results revealed no positive correlation between aBL and single stressors, indicating that aBL’s effectiveness is due to the simultaneous generation of multiple stressors. This multifactorial effect suggests that aBL targets microbial cells more precisely than single stressors such as hydrogen peroxide. No single gene knockout conferred specific resistance, highlighting aBL’s potential as an antimicrobial strategy.

Antimicrobial blue light (aBL) is emerging as a promising non-pharmacological antimicrobial strategy with multitarget activity and a low risk of resistance development. Its effects are primarily mediated by endogenous porphyrins, but additional chromophores such as flavins and bacterial pigments can also contribute. In this review, we summarize current evidence on these photosensitizers and discuss how their diversity underlies species-dependent susceptibility, with special emphasis on the variable responses of Streptococcus and Enterococcus spp. At the molecular level, aBL-induced reactive oxygen species (ROS) damage DNA, proteins, and membranes, disrupting metabolic pathways, efflux pumps, and membrane integrity. Genetic determinants, including porphyrin biosynthesis genes, cell envelope regulators, and components of the SOS and oxidative stress responses, further influence susceptibility. Transcriptomic studies reveal global shifts affecting stress responses, energy metabolism, motility, membrane remodeling, metal ion regulation, and virulence-associated functions. Additional microbial determinants, such as biofilm formation, virulence factors, and antibiotic resistance profiles, modulate treatment outcomes, while environmental and physiological parameters (growth phase, inoculum density, temperature, wavelength, and irradiance) strongly impact efficacy. Altogether, these findings indicate that aBL efficacy is shaped by a complex interplay of chromophore availability, genetic background, physiological state, and external conditions. Clarifying these mechanisms is essential to optimize aBL for clinical and industrial applications. In summary, this review provides a comprehensive overview of the molecular and physiological bases of bacterial susceptibility to aBL and outlines key gaps that must be addressed to translate this modality into routine antimicrobial practice.