Due to rapidly growing antimicrobial resistance, there is an urgent need to develop alternative, non-antibiotic strategies. Recently, numerous light-based approaches, demonstrating killing efficacy regardless of microbial drug resistance, have gained wide attention and are considered some of the most promising antimicrobial modalities. These light-based therapies include five treatments for which high bactericidal activity was demonstrated using numerous in vitro and in vivo studies: antimicrobial blue light (aBL), antimicrobial photodynamic inactivation (aPDI), pulsed light (PL), cold atmospheric plasma (CAP), and ultraviolet (UV) light. Based on their multitarget activity leading to deleterious effects to numerous cell structures—i.e., cell envelopes, proteins, lipids, and genetic material—light-based treatments are considered to have a low risk for the development of tolerance and/or resistance. Nevertheless, the most recent studies indicate that repetitive sublethal phototreatment may provoke tolerance development, but there is no standard methodology for the proper evaluation of this phenomenon. The statement concerning the lack of development of resistance to these modalities seem to be justified; however, the most significant motivation for this review paper was to critically discuss existing dogma concerning the lack of tolerance development, indicating that its assessment is more complex and requires better terminology and methodology.

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.

The global challenge of antimicrobial resistance (AMR) has been framed primarily in terms of genetic resistance mechanisms. Nevertheless, bacteria can also survive antimicrobial stress through phenotypic plasticity, resulting in transient, non-genetic states such as tolerance, persistence, and population-level resilience. These phenotypic states complicate diagnostic efforts, diminish antibiotic efficacy, and contribute to the chronic nature of infections even in the absence of heritable resistance. This review evaluates phenotypic plasticity as a significant yet underrecognized factor in AMR, with a focus on responses to oxidative and photodynamic stress. Key manifestations of plasticity are discussed, including morphological and metabolic remodeling such as filamentation, small-colony variants, and metabolic rewiring, as well as envelope- and biofilm-associated heterogeneity and regulatory flexibility mediated by gene networks and horizontal regulatory transfer. The review highlights plastic responses elicited by reactive oxygen species-mediated stress and antimicrobial photodynamic inactivation, where single-cell heterogeneity, biofilm and mucus barriers, and light-dependent cues influence bacterial survival. Case studies are presented to demonstrate how photodynamic strategies can induce transient protective states and act synergistically with antibiotics, revealing mechanisms of action that extend beyond conventional single-target therapeutic models. Drawing on evidence from single-cell analyses, biofilm ecology, and experimental evolution, this review establishes phenotypic plasticity as a central element in the chemical biology of AMR. Enhanced understanding of plasticity is essential for advancing diagnostics, informing the development of adjuvant therapies, and predicting bacterial responses to novel antimicrobial interventions.

The increasing reliance on light-based antimicrobial technologies, such as antimicrobial blue light (aBL) and antimicrobial photodynamic inactivation (aPDI), underscores the urgent need to comprehend bacterial survival strategies beyond conventional resistance. Two key phenotypes—tolerance and resilience—have emerged as critical but often conflated mechanisms by which bacteria withstand oxidative and photodynamic stress. While tolerance refers to delayed bacterial killing without changes in MIC, resilience encompasses the active restoration of cellular function after transient stress exposure. Both phenomena may impair treatment outcomes and contribute to long-term persistence, even in the absence of genetic resistance. This review dissects the molecular mechanisms underlying tolerance and resilience, with a focus on their relevance to bacterial responses to reactive oxygen species generated by light-based or chemical stressors. The regulatory and effector overlap between these phenotypes is examined, including antioxidant defense systems, DNA repair pathways, and metabolic rewiring. Furthermore, the role of phenotypic heterogeneity and cross-stress protection in blurring the boundary between survival and recovery is discussed, highlighting challenges in experimental interpretation. Finally, the implications of these adaptive strategies are evaluated in the context of antimicrobial efficacy and safety, with an emphasis on kinetic assays and multidimensional profiling as tools to capture complex treatment outcomes. Clarifying the distinction between tolerance and resilience may help guide the development of robust and evolutionarily stable antimicrobial phototherapies.

Antimicrobial blue light (aBL) is a promising non-antibiotic approach to fighting multidrug-resistant bacteria. However, the complete mechanism of aBL action is not fully understood yet. This study contributes to a better understanding that the response to aBL depends on many factors and that it is hardly possible to identify a predominant mechanism underlying microbial sensitivity to photoinactivation. The results of this study provide insights into genetic changes that may lead to bacterial survival at higher aBL doses, giving rise to aBL-resistant strains. To our best knowledge, this is the first study concerning genome-wide mutant testing of aBL. We managed to identify 64 single-gene mutants that lacked certain protective genes expressing aBL-increased sensitivity. IMPORTANCE: Increasing antibiotic resistance and the lack of new antibiotic-like compounds to combat bacterial resistance are significant problems of modern medicine. The development of new alternative therapeutic strategies is extremely important. Antimicrobial blue light (aBL) is an innovative approach to combat multidrug-resistant microorganisms. aBL has a multitarget mode of action; however, the full mechanism of aBL antibacterial action requires further investigation. In addition, the potential risk of resistance development to this treatment should be considered.

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.