Breaking down antibiotic resistance in methicillin-resistant Staphylococcus aureus: Combining antimicrobial photodynamic and antibiotic treatments

The widespread use of antibiotics drives the evolution of antimicrobial-resistant bacteria (ARB), threatening patients and healthcare professionals. Therefore, the development of novel strategies to combat resistance is recognized as a global healthcare priority. The two methods to combat ARB are development of new antibiotics or reduction in existing resistances. Development of novel antibiotics is a laborious and slow-progressing task that is no longer a safe reserve against looming risks. In this research, we suggest a method for reducing resistance to extend the efficacious lifetime of current antibiotics. Antimicrobial photodynamic therapy (aPDT) is used to generate reactive oxygen species (ROS) via the photoactivation of a photosensitizer. ROS then nonspecifically damage cellular components, leading to general impairment and cell death. Here, we test the hypothesis that concurrent treatment of bacteria with antibiotics and aPDT achieves an additive effect in the elimination of ARB. Performing aPDT with the photosensitizer methylene blue in combination with antibiotics chloramphenicol and tetracycline results in significant reductions in resistance for two methicillin-resistant Staphylococcus aureus (MRSA) strains, USA300 and RN4220. Additional resistant S. aureus strain and antibiotic combinations reveal similar results. Taken together, these results suggest that concurrent aPDT consistently decreases S. aureus resistance by improving susceptibility to antibiotic treatment. In turn, this development exhibits an alternative to overcome some of the growing MRSA challenge.

Antimicrobial-resistant bacteria (ARB) have raised public health concerns since the beginning of industrial antibiotic production in the 1940s (1). Healthcare-associated infections with ARB have caused significant morbidity, mortality, and economic burdens (2). In fact, according to the Centers for Disease Control and Prevention, over 2.8 million ARB infections occur every year, resulting in more than 35,000 deaths in total (3). Although many bacterial pathogens are successfully treated with antibiotic therapies, the treatment itself is the leading source of increasing antimicrobial resistance (4). The treatment methods for ARB most commonly involve the use of combination antibiotic therapies or treatment with adjuvants that target bacterial resistance mechanisms, including efflux pumps (5). These treatments are primarily beneficial due to their specific effectiveness against antimicrobial-resistant organisms but include moderate to severe undesired side effects such as neurotoxicity, kidney damage, and myelosuppression (6–8). Those who acquire ARB infections are much more likely to develop severe symptoms leading to poorer outcomes than those who acquire non-ARB infections (9). Some levels of prevalence and transmission dynamics are understood and being explored by improved monitoring, such as the World Health Organization global antimicrobial resistance and use surveillance system, which aid proper type and deployment of preventative measures. Nonetheless, a long-term solution to infection with diverse ARB has yet to be identified and serves as the ultimate goal.Antimicrobial photodynamic therapy (aPDT) is a technique by which pathogens are inactivated by reactive oxygen species (ROS) generated from the coincidence of molecular oxygen, a photosensitizer, and characteristic light of a particular wavelength (10). The resulting ROS may cause nonspecific biological oxidative stress (11). Additionally, noncytotoxic photosensitizers have been shown to influence specific drug localization and photoactivation wavelengths, corresponding to control of aPDT activation depth in tissues (12). The absorption spectrum of methylene blue, with a maximum molar absorptivity of 85,000 M⁻¹ cm⁻¹ at 664 nm, is concentration-dependent to dimerization and proportional to ionic strength at interfaces. The quantum yield of methylene blue fluorescence is dependent on the solvent used, and its interactions display a low quantum yield (0.04) in water. However, the fluorescence signal of methylene blue is concentration- and aggregation-dependent, and reductions are observed when the molecule interacts with membranes, proteins, and other biological substrates that favor electron transfer reactions. Methylene blue can act in both type I and type II mechanisms depending on its aggregation state. Methylene blue undergoes reduction reactions after electronic excitation, generating semireduced radicals and promoting mitochondrial NAD(P)H oxidation. Leuco-methylene blue generates high proton potentials, resulting in the generation of half of the 1O² radical species. The type II mechanism is favored in biological systems with higher oxygen concentrations in membranes than in water. The highest affinity of the molecule is for negatively charged interfaces, and melanin has been described in studies that illustrate methylene blue applications in dermatology. However, its quantum efficiency is reduced in tumors when administered intravenously (13).aPDT treatments have demonstrated efficacy against numerous microorganisms in experimental animal models. Although many photosensitizers are FDA approved for other photodynamic applications such as cancer therapy, they have not been approved for aPDT applications against ARBs (14). Methylene blue biocompatibility is exploited for the treatment of methemoglobinemia. Membranes of diseased tissues have redox properties that facilitate methylene blue reduction or oxidation. Methylene blue can also easily permeate membranes through lipid bilayers (15).Nevertheless, the promise of the approach has been demonstrated. For example, reports have shown that moderate aPDT doses eliminated most methicillin-resistant Staphylococcus aureus (MRSA) (16). Despite these advances, whether aPDT can offer potential benefits by reducing the doses required to combat ARB infection remains unresolved. If this were the case, the benefits associated with reduced antibiotic-associated side effects would be substantial. The radiation wavelength is normally chosen as the absorption peak of the molecule; this is the wavelength region of the most significant conversion of light energy into electronic energy of the molecule. Being electronically excited, the molecules are able, by collisional energy transfer, to produce the maximum number of ROS, thus maximizing the reactive effect of the photodynamic action. During clinical applications of aPDT, photosensitizers are usually administered systemically or topically. They are then activated by penetrating light greater than 600 nm in the therapeutic window of 600 to 800 nm (17). Wavelengths shorter than 600 nm are absorbed by hemoglobin, and wavelengths longer than 950 nm are absorbed by biological molecules that exhibit water vibration.In this study, we tested the hypothesis that aPDT improves antibiotic treatment effectiveness against MRSA. We found that aPDT did indeed improve the efficacy of antibiotic treatment against clinical isolates of MRSA. Taken together, these data set the stage for further development of aPDT as a strategy for combatting ARB infections.