Effective Treatment of Cutaneous Mold Infections by Antimicrobial Blue Light That Is Potentiated by Quinine.

BACKGROUND: Cutaneous mold infections commonly result from an array of traumatic injuries that involve direct inoculation of contaminated soil into wounds. Here, we explored the use of antimicrobial blue light (aBL; 405 nm wavelength) and the combination of aBL with quinine hydrochloride (aBL + Q-HCL) for the treatment of cutaneous mold infections. METHODS: Efficacy of aBL and aBL + Q-HCL in killing clinically important pathogenic molds (Aspergillus fumigatus, Aspergillus flavus, and Fusarium oxyprorum) was investigated. Ultraperformance liquid chromatography identified and quantified endogenous porphyrins in the mold conidia. Finally, a mouse model of dermabrasion wound infected with a bioluminescent variant of A. fumigatus was developed to investigate the efficacy of aBL in treating cutaneous mold infections. RESULTS: We demonstrated that mold conidia are tolerant to aBL, but Q-HCL enhances efficacy. Transmission electron microscopy revealed intracellular damage by aBL. aBL + Q-HCL resulted in intracellular and cell wall damage. Porphyrins were observed in all mold strains, with A. fumigatus having the highest concentration. aBL and aBL + Q-HCL effectively reduced the burden of A. fumigatus within an established dermabrasion infection and limited recurrence posttreatment. CONCLUSIONS: aBL and aBL + Q-HCL may offer a novel approach for the treatment of mold infections.

Antimicrobial blue light inactivation of pathogenic microbes: State of the art.

As an innovative non-antibiotic approach, antimicrobial blue light in the spectrum of 400-470nm has demonstrated its intrinsic antimicrobial properties resulting from the presence of endogenous photosensitizing chromophores in pathogenic microbes and, subsequently, its promise as a counteracter of antibiotic resistance. Since we published our last review of antimicrobial blue light in 2012, there have been a substantial number of new studies reported in this area. Here we provide an updated overview of the findings from the new studies over the past 5 years, including the efficacy of antimicrobial blue light inactivation of different microbes, its mechanism of action, synergism of antimicrobial blue light with other angents, its effect on host cells and tissues, the potential development of resistance to antimicrobial blue light by microbes, and a novel interstitial delivery approach of antimicrobial blue light. The potential new applications of antimicrobial blue light are also discussed.

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.

In vitro activity of Melaleuca alternifolia (tea tree) oil on filamentous fungi and toxicity to human cells.

Invasive fungal wound infections (IFIs) are increasingly reported in trauma patients and cause considerable morbidity and mortality despite standard of care treatment in trauma centers by experienced medical personnel. Topical agents such as oil of melaleuca, also known as tea tree oil (TTO), have been proposed for adjunctive treatment of IFIs. We evaluated the activity of TTO against filamentous fungi associated with IFIs by testing 13 clinical isolates representing nine species via time-kill assay with seven concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, and 1%). To ascertain the safety of topical application to wounds, cell viability assays were performed in vitro using human fibroblasts, keratinocytes, osteoblasts, and umbilical vein endothelial cells with 10 concentrations of TTO (75%, 50%, 25%, 10%, 5%, and 10-fold serial dilutions from 1 to 0.0001%) at five time points (5, 15, 30, 60, and 180 min). Compatibility of TTO with explanted porcine tissues was also assessed with eight concentrations of TTO (100%, 75%, 50%, 25%, 10%, 5%, 1%, and 0.1%) at the time points used for cellular assays and at 24 h. The time-kill studies showed that fungicidal activity was variable between isolates. The effect of TTO on cell viability was primarily concentration dependent with significant cytotoxicity at concentrations of ≥ 10% and ≥ 50% for cells lines and whole tissue, respectively. Our findings demonstrate that TTO possesses antifungal activity against filamentous fungi associated with IFIs; furthermore that negligible effects on whole tissues, in contrast to individual cells, were observed following exposure to TTO. Collectively, these findings indicate a potential use of TTO as topical treatment of IFIs.

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).

Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model.

This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(dl-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin for up to 8 weeks. Three formulations with either burst release or extended release at different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2×10(7) colony forming units ml(-1)). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks on culture of the defect, saliva, or blood. Defects with high dose extended release implants had greater soft tissue healing compared with defects with burst release implants, with 8 of 10 animals showing healed mucosae compared with 2 of 10 respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing compared with implants with burst release of colistin from a gelatin carrier.

Blue light eliminates community-acquired methicillin-resistant Staphylococcus aureus in infected mouse skin abrasions.

BACKGROUND AND OBJECTIVE: Bacterial skin and soft tissue infections (SSTI) affect millions of individuals annually in the United States. Treatment of SSTI has been significantly complicated by the increasing emergence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains. The objective of this study was to demonstrate the efficacy of blue light (415 ± 10 nm) therapy for eliminating CA-MRSA infections in skin abrasions of mice. METHODS: The susceptibilities of a CA-MRSA strain (USA300LAC) and human keratinocytes (HaCaT) to blue light inactivation were compared by in vitro culture studies. A mouse model of skin abrasion infection was developed using bioluminescent USA300LAC::lux. Blue light was delivered to the infected mouse skin abrasions at 30 min (acute) and 24 h (established) after the bacterial inoculation. Bioluminescence imaging was used to monitor in real time the extent of infection in mice. RESULTS: USA300LAC was much more susceptible to blue light inactivation than HaCaT cells (p=0.038). Approximately 4.75-log10 bacterial inactivation was achieved after 170 J/cm(2) blue light had been delivered, but only 0.29 log10 loss of viability in HaCaT cells was observed. Transmission electron microscopy imaging of USA300LAC cells exposed to blue light exhibited disruption of the cytoplasmic content, disruption of cell walls, and cell debris. In vivo studies showed that blue light rapidly reduced the bacterial burden in both acute and established CA-MRSA infections. More than 2-log10 reduction of bacterial luminescence in the mouse skin abrasions was achieved when 41.4 (day 0) and 108 J/cm(2) (day 1) blue light had been delivered. Bacterial regrowth was observed in the mouse wounds at 24 h after the blue light therapy. CONCLUSIONS: There exists a therapeutic window of blue light for bacterial infections where bacteria are selectively inactivated by blue light while host tissue cells are preserved. Blue light therapy has the potential to rapidly reduce the bacterial load in SSTI.

Blue light rescues mice from potentially fatal Pseudomonas aeruginosa burn infection: efficacy, safety, and mechanism of action.

Blue light has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. However, the use of blue light for wound infections has not been established yet. In this study, we demonstrated the efficacy of blue light at 415 nm for the treatment of acute, potentially lethal Pseudomonas aeruginosa burn infections in mice. Our in vitro studies demonstrated that the inactivation rate of P. aeruginosa cells by blue light was approximately 35-fold higher than that of keratinocytes (P = 0.0014). Transmission electron microscopy revealed blue light-mediated intracellular damage to P. aeruginosa cells. Fluorescence spectroscopy suggested that coproporphyrin III and/or uroporphyrin III are possibly the intracellular photosensitive chromophores associated with the blue light inactivation of P. aeruginosa. In vivo studies using an in vivo bioluminescence imaging technique and an area-under-the-bioluminescence-time-curve (AUBC) analysis showed that a single exposure of blue light at 55.8 J/cm(2), applied 30 min after bacterial inoculation to the infected mouse burns, reduced the AUBC by approximately 100-fold in comparison with untreated and infected mouse burns (P < 0.0001). Histological analyses and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays indicated no significant damage in the mouse skin exposed to blue light at the effective antimicrobial dose. Survival analyses revealed that blue light increased the survival rate of the infected mice from 18.2% to 100% (P < 0.0001). In conclusion, blue light therapy might offer an effective and safe alternative to conventional antimicrobial therapy for P. aeruginosa burn infections.

Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and beyond?

Blue light, particularly in the wavelength range of 405-470 nm, has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. In addition, it is commonly accepted that blue light is much less detrimental to mammalian cells than ultraviolet irradiation, which is another light-based antimicrobial approach being investigated. In this review, we discussed the blue light sensing systems in microbial cells, antimicrobial efficacy of blue light, the mechanism of antimicrobial effect of blue light, the effects of blue light on mammalian cells, and the effects of blue light on wound healing. It has been reported that blue light can regulate multi-cellular behavior involving cell-to-cell communication via blue light receptors in bacteria, and inhibit biofilm formation and subsequently potentiate light inactivation. At higher radiant exposures, blue light exhibits a broad-spectrum antimicrobial effect against both Gram-positive and Gram-negative bacteria. Blue light therapy is a clinically accepted approach for Propionibacterium acnes infections. Clinical trials have also been conducted to investigate the use of blue light for Helicobacter pylori stomach infections and have shown promising results. Studies on blue light inactivation of important wound pathogenic bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa have also been reported. The mechanism of blue light inactivation of P. acnes, H. pylori, and some oral bacteria is proved to be the photo-excitation of intracellular porphyrins and the subsequent production of cytotoxic reactive oxygen species. Although it may be the case that the mechanism of blue light inactivation of wound pathogens (e.g., S. aureus, P. aeruginosa) is the same as that of P. acnes, this hypothesis has not been rigorously tested. Limited and discordant results have been reported regarding the effects of blue light on mammalian cells and wound healing. Under certain wavelengths and radiant exposures, blue light may cause cell dysfunction by the photo-excitation of blue light sensitizing chromophores, including flavins and cytochromes, within mitochondria or/and peroxisomes. Further studies should be performed to optimize the optical parameters (e.g., wavelength, radiant exposure) to ensure effective and safe blue light therapies for infectious disease. In addition, studies are also needed to verify the lack of development of microbial resistance to blue light.

In vitro and in vivo activity of first generation cephalosporins against Leptospira.

Third generation cephalosporins are commonly used in the treatment of leptospirosis. The efficacy of first generation cephalosporins has been less well-studied. Susceptibility testing of 13 Leptospira strains (11 serovars) to cefazolin and cephalexin was conducted using broth microdilution. Median minimal inhibitory concentration (MIC) for cefazolin and cephalexin ranged from < 0.016 to 2 µg/mL (MIC(90) = 0.5 µg/mL) and from 1 to 8 µg/mL (MIC(90) = 8 µg/mL), respectively. Efficacy of cefazolin and cephalexin in an acute lethal hamster model of leptospirosis was studied. Survival rates for cefazolin were 80%, 100%, and 100%, and survival rates for cephalexin were 50%, 80%, and 100% (treated with 5, 25, and 50 mg/kg per day for 5 days, respectively). Each treatment group showed improved survival compared with no treatment (P < 0.01), and none of the therapies, regardless of dose, was statistically significantly different than doxycycline. These results support a potential role for first generation cephalosporins as alternative therapies for leptospirosis.

Colistin heteroresistance in acinetobacter and its association with previous colistin therapy.

Colistin heteroresistance has been reported among Acinetobacter isolates; however, its association with prior colistin therapy has not been not described. A population analysis profile identified resistant Acinetobacter subpopulations from colistin-susceptible clinical isolates. The proportion of cells exhibiting heteroresistance was significantly higher among isolates recovered from patients treated with colistin.

Effect of timing and duration of azithromycin therapy of leptospirosis in a hamster model.

OBJECTIVES: Azithromycin is not associated with significant adverse effects or restricted usage in certain populations unlike standard antileptospirosis agents. In this study, the utility of short courses of azithromycin in treating or preventing leptospirosis was investigated in a lethal hamster model. METHODS: All hamsters were infected intraperitoneally with 10(5) leptospires. In experiment one, animals received 5 mg/kg of doxycycline or 10 mg/kg of azithromycin via intraperitoneal injection beginning on the second day after infection and continuing once daily for 1, 2, 3 or 5 days. In experiment two, animals received 1 or 2 day courses of azithromycin initiated 2 or 4 days following infection, or 4 days prior to infection. RESULTS: All untreated control animals died between the sixth and ninth day following infection. In experiment one, survival rates in the doxycycline groups were 0, 50, 80 and 100% for those animals treated for 1, 2, 3 and 5 days, respectively. Except for the 1 day treatment group (which had an 80% survival), there was 100% survival in all azithromycin-treated groups. In experiment two, all animals treated after infection survived until study completion. No animals survived with 1 day of therapy started 4 days prior to infection while only 20% survived if they received 2 days. CONCLUSIONS: These results suggest short-course therapy with azithromycin, even started well after infection, is efficacious in preventing mortality from acute leptospirosis.

Antimicrobial therapy of leptospirosis.

PURPOSE OF REVIEW: Leptospirosis is an important but often overlooked zoonotic disease that can cause significant morbidity and mortality. The optimal antimicrobial treatment for this disease has not been established. This review summarizes the most recent literature pertaining to the use of antimicrobial agents in the treatment of leptospirosis. RECENT FINDINGS: Leptospira are highly susceptible to a wide variety of antimicrobials in vitro. Despite this, it is not clear what the best choice of antimicrobial agents is for human disease. Based on the best available literature, the current choices of treatment for leptospirosis include penicillin, doxycycline, cefotaxime, ceftriaxone and azithromycin. Penicillin has long been considered the treatment of choice. Doxycycline is a reasonable alternative, but concerns exist regarding its use in all patients. Recent trials have demonstrated that cefotaxime and ceftriaxone are also acceptable agents. For a variety of reasons, these may be the preferred agents at this time. Azithromycin appears promising for the treatment of less severe disease. Another option for treating leptospirosis is the fluoroquinolone antimicrobials, although adequate human trials are lacking to fully support their use. SUMMARY: Leptospirosis is an important cause of morbidity and mortality worldwide. Despite this, the optimal treatment is not fully defined.

Treatment of multidrug resistant Acinetobacter.

PURPOSE OF REVIEW: Acinetobacter baumannii-calcoaceticus complex has become a serious nosocomial pathogen due to its persistence in the hospital environment and its broad antimicrobial resistance patterns. This review summarizes the most recent literature pertaining to the clinical management of infections with this bacteria emphasizing in-vitro antimicrobial resistance patterns and antimicrobial efficacy in animals and humans. RECENT FINDINGS: Although this pathogen can be associated with an elevated crude mortality, it only contributes to this mortality in a subset of high-risk patients. Determining in-vitro activity of antimicrobial agents can be problematic due to conflicting results sometimes obtained through different testing methods. There is no simple answer as to the most appropriate antimicrobial therapy secondary to lack of adequate studies. Imipenem/cilastatin, amikacin, ampicillin/sulbactam, colistin, rifampin, and tetracyclines are typically active against these bacteria. It is also not clear if combination therapy is more effective than monotherapy. In cases in which A. baumannii-calcoaceticus complex bacteria are resistant to all available agents, we have prolonged infusion times, increased drug dose, and altered route of instillation, such as nebulized therapy for pulmonary infections with mixed results. A primary goal of A. baumannii-calcoaceticus complex management should be to prevent initial colonization and subsequent infection by adequate infection control. SUMMARY: The A. baumannii-calcoaceticus complex continues to play a significant role in our healthcare systems. Prompt and adequate therapy with agents having in-vitro activity is required once it is established that the bacteria represents infection and not colonization. Aggressive infection control policies should be enforced when this pathogen is identified.

The role of antifungal susceptibility testing in the therapy of candidiasis.

Prior to the introduction of azoles, no real need for antifungal susceptibility testing (AFST) existed, as amphotericin B was the only agent available to treat systemic candidiasis. Introduction of fluconazole and itraconazole provided alternate, less toxic antifungal therapies. Intrinsic resistance of Candida krusei, decreased susceptibility of Candida glabrata, and development of resistance by Candida albicans (in mucosal disease in AIDS) to azoles led to development of our current AFST methodologies. The goal of AFST, like that of antibacterial susceptibility testing, is to predict clinical response, or at least to forecast failure. Although the ability of AFST to predict clinical outcome (clinical correlation) is still being fully elucidated, current methodologies do appear to reliably predict clinical resistance to azoles. Ready access to AFST is currently limited, affecting its timely use, but even with this lack of timeliness, AFST can still play an important role in patient care. Important potential roles include: 1) use in the development of local antibiograms to aid empiric selection of antifungals; 2) testing of isolates from candidemia or deep infection to aid in selection of long-term therapies; and, 3) the testing of isolates from recurrent mucosal disease to aid in selection of alternative regimens.