Thermo-responsive chitosan hydrogel for healing of full-thickness wounds infected with XDR bacteria isolated from burn patients: In vitro and in vivo animal model.

Treatment of non-healing skin wounds infected with extensively drug-resistant (XDR) bacteria remains as a big challenge. To date, different biomaterials have been applied for treatment of post-wound infections, nevertheless their efficacy for treatment of the wounds infected with XDR isolates has not been determined yet. In this study, the potential of the thermo-responsive chitosan (TCTS) hydrogel for protection of full-thickness wounds XDR bacteria isolated from burn patients was evaluated both in vitro and in vivo in a rat model. Antibacterial activity of the TCTS hydrogel against standard strain and clinical isolates of Acinetobacter baumannii, cytobiocompatibility for Hu02 fibroblast cells, degradation rate and swelling ratio were determined in vitro. MTT assay and disk diffusion test indicated no detectable cytotoxicity and antibacterial activity in vitro, respectively. In vivo study showed significant acceleration of wound healing, re-epithelialization, wound closure, and decreased colony count in the TCTS hydrogel group compared with control. This study suggests TCTS hydrogel as an excellent wound dressing for management of the wounds infected with XDR bacteria, and now promises to proceed with clinical investigations.

Antibiotics Act with vB_AbaP_AGC01 Phage against Acinetobacter baumannii in Human Heat-Inactivated Plasma Blood and Galleria mellonella Models.

Increasing multidrug resistance has led to renewed interest in phage-based therapy. A combination of the bacteriophages and antibiotics presents a promising approach enhancing the phage therapy effectiveness. First, phage candidates for therapy should be deeply characterized. Here we characterize the bacteriophage vB_AbaP_AGC01 that poses antibacterial activity against clinical Acinetobacter baumannii strains. Moreover, besides genomic and phenotypic analysis our study aims to analyze phage-antibiotic combination effectiveness with the use of ex vivo and in vivo models. The phage AGC01 efficiently adsorbs to A. baumannii cells and possesses a bacteriolytic lifecycle resulting in high production of progeny phages (317 ± 20 PFU × cell-1). The broad host range (50.27%, 93 out of 185 strains) against A. baumannii isolates and the inability of AGC01 to infect other bacterial species show its high specificity. Genomic analysis revealed a high similarity of the AGC01 genome sequence with that of the Friunavirus genus from a subfamily of Autographivirinae. The AGC01 is able to significantly reduce the A. baumannii cell count in a human heat-inactivated plasma blood model (HIP-B), both alone and in combination with antibiotics (gentamicin (GEN), ciprofloxacin (CIP), and meropenem (MER)). The synergistic action was observed when a combination of phage treatment with CIP or MER was used. The antimicrobial activity of AGC01 and phage-antibiotic combinations was confirmed using an in vivo larva model. This study shows the greatest increase in survival of G. mellonella larvae when the combination of phage (MOI = 1) and MER was used, which increased larval survival from 35% to 77%. Hence, AGC01 represents a novel candidate for phage therapy. Additionally, our study suggests that phages and antibiotics can act synergistically for greater antimicrobial effect when used as combination therapy.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria. An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process. Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells. In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

Intranasal treatment with bacteriophage rescues mice from Acinetobacter baumannii-mediated pneumonia.

AIM: With the emergence of drug-resistant bacteria, finding alternative agents to treat antibiotic-resistant bacterial infections is imperative. MATERIALS & METHODS: A mouse pneumonia model was developed by combining cyclophosphamide pretreatment and Acinetobacter baumannii challenge, and a lytic bacteriophage was evaluated for its therapeutic efficacy in this model by examining the survival rate, bacterial load in the lung and lung pathology. RESULTS: Intranasal instillation with bacteriophage rescued 100% of mice following lethal challenge with A. baumannii. Phage treatment reduced bacterial load in the lung. Microcomputed tomography indicated a reduction in lung inflammation in mice given phage. CONCLUSION: This research demonstrates that intranasal application of bacteriophage is viable, and could provide complete protection from pneumonia caused by A. baumannii.

Pulsed electric fields for burn wound disinfection in a murine model.

Emerging bacterial resistance renders many antibiotics ineffective, making alternative strategies of wound disinfection important. Here the authors report on a new, physical burn wound disinfection method: pulsed electric fields (PEFs). High voltage, short PEFs create nonthermal, permanent damage to cell membranes, possibly by irreversible electroporation. In medicine, PEF technology has recently been used for nonthermal ablation of solid tumors. The authors have expanded the spectrum of PEF applications in medicine to burn wound disinfection. A third-degree burn was induced on the dorsal skin of C57BL/6 mice. Immediately after the injury, the burn wound was infected with Acinetobacter baumannii expressing the luxCDABE operon. Thirty minutes after infection, the infected areas were treated with 80 pulses delivered at 500 V/mm, 70 µs, 1 Hz. The authors used bioluminescence to quantify bacteria on skin. Three animals were used for each experimental condition. PEFs were effective in the disinfection of infected burned murine skin. The bacterial load reduction correlated with the number of delivered pulses. Forty pulses of 500 V/mm led to a 2.04 ± 0.29 Log10 reduction in bacterial load; 80 pulses led to the immediate 5.53 ± 0.30 Log10 reduction. Three hours after PEF, the bacterial reduction of the skin treated with 500 V/mm, 80 pulses was 4.91 ± 0.71 Log10. The authors introduce a new method of wound disinfection using high voltage, short PEFs. They believe that PEF technology may represent an important alternative to antibiotics in addressing bacterial contamination of wounds, particularly those contaminated with multidrug-resistant bacteria.

Antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model: implications for prophylaxis and treatment of combat-related wound infections.

In this study, we investigated the utility of antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model. A bioluminescent clinical isolate of multidrug-resistant A. baumannii was obtained. The susceptibility of A. baumannii to blue light (415 nm)-inactivation was compared in vitro to that of human keratinocytes. Repeated cycles of sublethal inactivation of bacterial by blue light were performed to investigate the potential resistance development of A. baumannii to blue light. A mouse model of third degree burn infected with A. baumannii was developed. A single exposure of blue light was initiated 30 minutes after bacterial inoculation to inactivate A. baumannii in mouse burns. It was found that the multidrug-resistant A. baumannii strain was significantly more susceptible than keratinocytes to blue light inactivation. Transmission electron microscopy revealed blue light-induced ultrastructural damage in A. baumannii cells. Fluorescence spectroscopy suggested that endogenous porphyrins exist in A. baumannii cells. Blue light at an exposure of 55.8 J/cm(2) significantly reduced the bacterial burden in mouse burns. No resistance development to blue light inactivation was observed in A. baumannii after 10 cycles of sublethal inactivation of bacteria. No significant DNA damage was detected in mouse skin by means of a skin TUNEL assay after a blue light exposure of 195 J/cm(2).

Emerging therapies for multidrug resistant Acinetobacter baumannii.

The global emergence of multidrug resistant Acinetobacter baumannii has reduced the number of clinically available antibiotics that retain activity against this pathogen. For this reason, the development of novel prevention and treatment strategies for infections caused by A. baumannii is necessary. Several studies have begun to characterize nonantibiotic approaches that utilize novel mechanisms of action to achieve antibacterial activity. Recent advances in phage therapy, iron chelation therapy, antimicrobial peptides, prophylactic vaccination, photodynamic therapy, and nitric oxide (NO)-based therapies have all been shown to have activity against A. baumannii. However, before these approaches can be used clinically there are still limitations and remaining questions that must be addressed.

Isolation and characterization of φkm18p, a novel lytic phage with therapeutic potential against extensively drug resistant Acinetobacter baumannii.

AIMS: To isolate phages against extensively drug resistant Acinetobacter baumannii (XDRAB) and characterize the highest lytic capability phage as a model to evaluate the potential on phage therapy. METHODS AND RESULTS: Eight phages were isolated from hospital sewage and showed narrow host spectrum. Phage φkm18p was able to effectively lyse the most XDRAB. It has a dsDNA genome of 45 kb in size and hexagonal head of about 59 nm in diameter and no tail. Bacterial population decreased quickly from 10(8) CFU ml(-1) to 10(3) CFU ml(-1) in 30 min by φkm18p. The 185 kDa lysis protein encoded by φkm18p genome was detected when the extracted protein did not boil before SDS-PAGE; it showed that the lysis protein is a complex rather than a monomer. Phage φkm18p improved human lung epithelial cells survival rates when they were incubated with A. baumannii. Combination of phages (φkm18p, φTZ1 and φ314) as a cocktail could lyse all genotype-varying XDRAB isolates. CONCLUSION: Infections with XDRAB are extremely difficult to treat and development of a phage cocktails therapy could be a therapeutic alternative in the future. Phage φkm18p is a good candidate for inclusion in phage cocktails.

Novel therapies of multidrug-resistant Pseudomonas aeruginosa and Acinetobacter spp. infections: the state of the art.

Gram-negative non-fermenting bacilli, particularly Pseudomonas aeruginosa and Acinetobacter spp., are important opportunistic pathogens in hosppitalized patients, contributing to their morbidity and mortality. Recently, a rapid increase in frequency of multidrug-resistant clinical strains is being recorded, making the available therapeutic options very limited. Apart from the development of novel classes of antimicrobials, there is renewed interest in the use of old agents or new combinations of available drugs. Numerous in vitro investigations have been reported on the efficacy of different antimicrobials; however, they should be evaluated in experimental infection models and clinical trials. Novel approaches are being investigated, such as inhibition of virulence factor expression by pathogens or inhibition of their metabolic pathways. The use of bacteriophages, particularly those genetically modified, remains an alternative option in the therapy of infections caused by multidrug-resistant strains. Several vaccines against P. aeruginosa are under development. Apart from therapy with antimicrobial agents, eradication of outbreaks comprises implementation of strict infection control measures and prudent use of antimicrobials.

[The treatment of necrotizing otitis externa with a combination of surgery, antibiotics, specific immunoglobulins and hyperbaric oxygen therapy. Results of the Ulm Treatment Concept]. / Otitis externa necroticans. Kombinierter Einsatz von chirurgischer Therapie, Antibiose, spezifischen Immunglobulinen und hyperbarer Sauerstofftherapie--Ergebnisse des Ulmer Therapiekonzepts.

BACKGROUND: Even today, necrotizing otitis externa is still a life-threatening condition. The standard treatment concepts based on antibiotic therapy, topical treatment and--if necessary--surgical debridement of necrotic tissue, remain ineffective in many cases. In advanced stages, necrotizing otitis externa can therefore be expected to result in severe functional impairment or even a fatal outcome. PATIENTS AND METHODS: We describe our experience with a multimodal treatment concept that we have been using with great success at the Federal Armed Forces Hospital in Ulm since 1987. This treatment is based on four pillars: 1. Surgical debridement, 2. Use of a combination of antibiotics, and 3. Administration of specific immunoglobulins, accompanied by, 4. Hyperbaric oxygen therapy RESULTS: Over a period of more than 5 years 16 of the 22 patients treated on the basis of this multimodal concept remained free of recurrences. In addition, significant reductions in analgesic use and in the insulin doses needed by diabetic patients were possible. CONCLUSIONS: The treatment concept under scrutiny has proved particularly successful. The various modes involved and the results of this combined therapy are discussed.