Photodynamic Therapy for Acinetobacter baumannii Burn Infections in Mice

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 10⁸ 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.Acinetobacter baumannii is a gram-negative pathogenic bacterium that has recently attracted much attention due to its remarkable acquisition of multidrug resistance (18, 19). It has been reported to have caused intractable infections in traumatic wounds and burns suffered by military personnel injured in recent Middle Eastern conflicts (24, 25). Photodynamic therapy (PDT) is a rapidly expanding approach to the treatment of diseases because it eliminates unwanted cells, such as malignant cancer cells and infectious microbial cells. PDT involves the combination of nontoxic photosensitizers (PSs) and harmless visible light that, in the presence of oxygen, give reactive oxygen species by energy or electron transfer from the PS excited state that are able to oxidize biomolecules and thereby kill cells (17). The use of PDT to treat localized infections generally involves the topical application of a PS into the infected tissue, followed by illumination with red or near-infrared light that is able to penetrate the tissue (2, 9). Selectivity for bacteria over host tissue can be obtained by the appropriate chemical design of the PS to ensure that the molecule will preferentially bind to bacterial cells rather than mammalian cells. It has been determined by many researchers that the most important features of this molecular design are a combination of an overall cationic charge and water solubility (11, 16). Cationic charge is even more important in the case of gram-negative bacteria that possess a double membrane structure because that structure excludes many anionic and uncharged lipophilic molecules that can effectively penetrate gram-positive bacteria and fungal cells (14).We have previously reported that a covalent conjugate between the tetrapyrrole molecule known as chlorin(e6) (c_e6) and the synthetic cationic polymer polyethylenimine (PEI) is a highly effective PS with activity against gram-negative species (27).In this report we describe the establishment of a mouse model of A. baumannii infection in full-thickness thermal burns. We used a multidrug-resistant clinical isolate from a U.S. soldier that was transformed with a plasmid that encodes the entire lux operon from Photorhabdus luminescens and that allows bioluminescence imaging of the course of the infection noninvasively in real time. The topical application of PEI-c_e6 was carried out, followed by illumination with red light after 15 min.