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Background: Acinetobacter baumannii , a nosocomial pathogen, poses a major public health problem due to generating resistance to several antimicrobial agents. Antimicrobial photodynamic inactivation (APDI) employs a nontoxic dye as a photosensitizer (PS) and light to produce reactive oxygen species that destroy bacterial cells. The intracellular concentration of PS could be affected by factors such as the function of efflux pumps to emit PS from the cytosol. Objective: To evaluate the augmentation effect of an efflux pump inhibitor, verapamil, three multidrug-resistant A. baumannii were subjected to APDI by erythrosine B (EB). Methods and results: The combination of EB and verapamil along with irradiation at 530 nm induced a lethal effect and more than 3 log colony-forming unit reduction to all A. baumannii strains in planktonic state. In contrast, EB and irradiation alone could produce only a sublethal effect on two of the strains. Conclusions: These data suggest that verapamil increases the intracellular concentration of EB, which potentiates the lethal efficacy of APDI. Verapamil could be applied with EB and green light to improve their antimicrobial efficacy against A. baumannii -localized infections.

Photodynamic inactivation of bacteria (PIB) proves to be an additional method to kill pathogenic bacteria. PIB requires photosensitizer molecules that effectively generate reactive oxygen species like singlet oxygen when exposed to visible light. To allow a broad application in medicine, photosensitizers should be safe when applied in humans. Substances like vitamin B2, which are most likely safe, are known to produce singlet oxygen upon irradiation. In the present study, we added positive charges to flavin derivatives to enable attachment of these molecules to the negatively charged surface of bacteria. Two of the synthesized flavin derivatives showed a high quantum yield of singlet oxygen of approximately 75%. Multidrug resistant bacteria like MRSA (Methicillin resistant Staphylococcus aureus), EHEC (enterohemorrhagic Escherichia coli), Pseudomonas aeruginosa, and Acinetobacter baumannii were incubated with these flavin derivatives in vitro and were subsequently irradiated with visible light for seconds only. Singlet oxygen production in bacteria was proved by detecting its luminescence at 1270 nm. After irradiation, the number of viable bacteria decreased up to 6 log10 steps depending on the concentration of the flavin derivatives and the light dosimetry. The bactericidal effect of PIB was independent of the bacterial type and the corresponding antibiotic resistance pattern. In contrast, the photosensitizer concentration and light parameters used for bacteria killing did not affect cell viability of human keratinocytes (therapeutic window). Multiresistant bacteria can be safely and effectively killed by a combination of modified vitamin B2 molecules, oxygen and visible light, whereas normal skin cells survive. Further work will include these new photosensitizers for topical application to decolonize bacteria from skin and mucosa.

One environmental concern related to hospital effluents is discharge of them without preliminary treatment. Antimicrobial photodynamic inactivation (PDI) may represent an alternative to the traditional expensive, unsafe and not always effective disinfection methods. The main goal of this work was to assess the efficiency of PDI on clinical multidrug-resistant (MDR) bacteria in hospital wastewaters in order to evaluate its potential use in treating hospital effluents. The efficiency of PDI was assessed using a cationic porphyrin as the photosensitizer (PS), four MDR bacteria either in phosphate buffered saline or in filtrated hospital wastewaters. The synergistic effect of PDI and antibiotics (ampicillin and chloramphenicol) was also evaluated, as well as the effect of the surfactant sodium dodecyl sulfate (SDS). The results show the efficient inactivation of MDR bacteria in PBS (reduction of 6-8 log after 270 min of irradiation at 40 W m(-2) with 5.0 μM of PS). In wastewater, the inactivation of the four MDR bacteria was again efficient and the decrease in bacterial survival starts even sooner. A faster decrease in bacterial survival occurred when PDI was combined with the addition of antibiotics, at sub-inhibitory and inhibitory concentrations, but the SDS did not affect the PDI efficiency. It can be concluded that PDI has potential to be an effective alternative for the inactivation of MDR bacteria in hospital wastewaters and that the presence of antibiotics may enhance its effectiveness.

Background and Objective: Phenothiazinium dyes have been reported to be effective photosensitizers inactivating a wide range of microorganisms in vitro after illumination with red light. However, their application in vivo has not extensively been explored. This study evaluates the bactericidal activity of phenothiazinium dyes against multidrug-resistant Acinetobacter baumannii both in vitro and in vivo., Study Design/materials and Methods: We report the investigation of toluidine blue O, methylene blue, 1,9-dimethylmethylene blue, and new methylene blue for photodynamic inactivation of multidrug-resistant A. baumannii in vitro. The most effective dye was selected to carry out in vivo studies using third-degree mouse burns infected with a bioluminescent A. baumannii strain, upon irradiation with a 652 nm noncoherent light source. The mice were imaged daily for 2 weeks to observe differences in the bioluminescence-time curve between the photodynamic therapy (PDT)-treated mice in comparison with untreated burns., Results: All the dyes were effective in vitro against A. baumannii after 30 J/cm(2) irradiation of 635 or 652 nm red light had been delivered, with more effective killing when the dye remained in solution. New methylene blue was the most effective of the four dyes, achieving a 3.2-log reduction of the bacterial luminescence during PDT in vivo after 360 J/cm(2) and an 800 microM dye dose. Moreover, a statistically significant reduction of the area under the bioluminescence-time curve of PDT-treated mice was observed showing that the infection did not recur after PDT., Conclusions: Phenothiazinium dyes, and especially new methylene blue, are potential photosensitizers for PDT to treat burns infected with multidrug-resistant A. baumannii in vivo.

Multidrug-resistant Acinetobacter baumannii infections represent a growing problem, especially in traumatic wounds and burns suffered by military personnel injured in Middle Eastern conflicts. Effective treatment with traditional antibiotics can be extremely difficult, and new antimicrobial approaches are being investigated. One of these alternatives to antimicrobials could be the combination of nontoxic photosensitizers (PSs) and visible light, known as photodynamic therapy (PDT). We report on the establishment of a new mouse model of full-thickness thermal burns infected with a bioluminescent derivative of a clinical Iraqi isolate of A. baumannii and its PDT treatment by topical application of a PS produced by the covalent conjugation of chlorin(e6) to polyethylenimine, followed by illumination of the burn surface with red light. Application of 10(8) A. baumannii cells to the surface of 10-s burns made on the dorsal surface of shaved female BALB/c mice led to chronic infections that lasted, on average, 22 days and that were characterized by a remarkably stable bacterial bioluminescence. PDT carried out on day 0 soon after application of the bacteria gave over 3 log units of loss of bacterial luminescence in a light exposure-dependent manner, while PDT carried out on day 1 and day 2 gave an approximately 1.7-log reduction. The application of PS dissolved in 10% or 20% dimethyl sulfoxide without light gave only a modest reduction in the bacterial luminescence from mouse burns. Some bacterial regrowth in the treated burn was observed but was generally modest. It was also found that PDT did not lead to the inhibition of wound healing. The data suggest that PDT may be an effective new treatment for multidrug-resistant localized A. baumannii infections.