Assessing Biofilm Dispersal in Murine Wounds.

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Efficacy and safety of biofilm dispersal by glycoside hydrolases in wounds.

Novel anti-biofilm and dispersal agents are currently being investigated in an attempt to combat biofilm-associated wound infections. Glycoside hydrolases (GHs) are enzymes that hydrolyze the glycosidic bonds between sugars, such as those found within the exopolysaccharides of the biofilm matrix. Previous studies have shown that GHs can weaken the matrix, inducing bacterial dispersal, and improving antibiotic clearance. Yet, the number of GH enzymes that have been examined for potential therapeutic effects is limited. In this study, we screened sixteen GHs for their ability to disperse mono-microbial and polymicrobial biofilms grown in different environments. Six GHs, α-amylase (source: A. oryzae), alginate lyase (source: various algae), pectinase (source: Rhizopus sp.), amyloglucosidase (source: A. niger), inulinase (source: A. niger), and xylanase (source: A. oryzae), exhibited the highest dispersal efficacy in vitro. Two GHs, α-amylase (source: Bacillus sp.) and cellulase (source: A. niger), used in conjunction with meropenem demonstrated infection clearing ability in a mouse wound model. GHs were also effective in improving antibiotic clearance in diabetic mice. To examine their safety, we screened the GHs for toxicity in cell culture. Overall, there was an inverse relationship between enzyme exposure time and cellular toxicity, with twelve out of sixteen GHs demonstrating some level of toxicity in cell culture. However, only one GH exhibited harmful effects in mice. These results further support the ability of GHs to improve antibiotic clearance of biofilm-associated infections and help lay a foundation for establishing GHs as therapeutic agents for chronic wound infections.

Differential Efficacy of Glycoside Hydrolases to Disperse Biofilms.

Chronic wounds will impact 2% of the United States population at some point in their life. These wounds are often associated with a reoccurring, chronic infection caused by a community of microorganisms encased in an extracellular polymeric substance (EPS), or a biofilm. Biofilm-associated microbes can exhibit tolerance to antibiotics, which has prompted researchers to investigate therapeutics that improve antibiotic efficacy. Glycoside hydrolases (GHs), enzymes that target the polysaccharide linkages within the EPS, are one potential adjunctive therapy. In order to develop GH-based therapeutics, it is imperative that we understand whether the composition of biofilm EPS changes based on the environment and/or presence of other microbes. Here, we utilized α-amylase and cellulase to target the polysaccharides within the EPS of mono- and dual-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in three different models that vary in clinical relevancy. We show that biofilms established in an in vitro well-plate model are not strongly adhered to the polystyrene surface and do not accurately reflect the GH efficacy seen with biofilms grown in vivo. However, dispersal efficacy in an in vitro wound microcosm model was more reflective of that seen in a murine wound model. We also saw a striking loss of efficacy for cellulase to disperse S. aureus in both mono- and dual species biofilms grown in the wound models, suggesting that EPS constituents may be altered depending on the environment.

Utilizing glycoside hydrolases to improve the quantitation and visualization of biofilm bacteria.

The complexity of microbial biofilms offers several challenges to the use of traditional means of microbial research. In particular, it can be difficult to calculate accurate numbers of biofilm bacteria, because even after thorough homogenization or sonication, small pieces of the biofilm remain, which contain numerous bacterial cells and result in inaccurately low colony forming units (CFU). In addition, imaging of infected tissue ex vivo often results in a disparity between the CFU and the number of bacterial cells observed under the microscope. We hypothesized that this phenomenon is due to the biofilm extracellular polymeric substance decreasing the accessibility of stains and antibodies to the embedded bacterial cells. In this study, we describe incorporating EPS-degrading glycoside hydrolases for CFU determination to obtain a more accurate estimation of the viable cells and for immunohistochemistry to disrupt the biofilm matrix and increase primary antibody binding to the bacterial cells.

Nonselective NOS inhibition blunts the sweat response to exercise in a warm environment.

The role of nitric oxide synthase (NOS) inhibition in modulating human thermoregulatory control of sweating and cutaneous dilation was examined in 10 subjects (5 men and 5 women). Three intradermal microdialysis probes were placed in nonglabrous skin of the dorsum of the forearm. The control site was perfused with 0.9% saline, while the two remaining sites were perfused with a nonselective NOS inhibitor: 10 mM N(G)-nitro-L-arginine (L-NAME) or 10 mM N(G)-monomethyl-L-arginine (L-NMMA). Local sweat rate (SR) and skin blood flow (laser-Doppler velocimetry) were monitored directly over the path of the intradermal microdialysis probe while arterial blood pressure was measured in the opposite arm noninvasively. Thermoregulatory responses were induced by cycle ergometer exercise (60% peak oxygen consumption) in a warm environment (30 degrees C). Esophageal temperature increased 1.5 +/- 0.2 degrees C during the 30 min of exercise. The cutaneous dilator response between 5 and 30 min of exercise in the heat was attenuated by both 10 mM L-NAME and 10 mM L-NMMA (P < 0.05). However, 10 mM L-NAME was more effective in blunting the rise in cutaneous vascular conductance during exercise than L-NMMA (P < 0.05). NOS inhibition also reduced the rise in local SR between 10 and 30 min of exercise (P < 0.05). In this case, 10 mM L-NMMA was more effective in limiting the increase in local SR than 10 mM L-NAME (P < 0.05). We conclude that local production of nitric oxide in the skin or around the sweat gland augments local SR and cutaneous dilation during exercise in the heat.

Herpes Zoster Involving the Second Division of the Trigeminal Nerve: Case Report and Literature Review.

Herpes zoster along the maxillary division of the trigeminal nerve is a rare condition that is caused by reactivation of the varicella zoster virus that resides within the trigeminal ganglion after the primary infection of chickenpox. The disease may be manifested as a toothache during its prodromal stage. The active stage of the disease is characterized by the appearance of a vesicular rash. Postherpetic neuralgia is a common complication of herpes zoster after resolution of the facial and intraoral symptoms. There is increasing evidence for herpes zoster patients to develop stroke later in life. The present case reports the development of herpes zoster maxillaris in a 71-year-old man whose maxillary right canine was diagnosed as pulpal necrosis and symptomatic apical periodontitis and was subsequently treated endodontically by cleaning and shaping and filling the canal space with gutta-percha and an epoxy resin-based sealer. The patient presented 3 days later with midfacial ulceration, desquamation, and crusting as well as intraoral ulceration along the course of the V2 dermatome. After successful treatment with antiviral medication, postherpetic neuralgia developed within the next 2 months. Complete resolution of the neuralgia occurred at the 4-month recall with negligible facial scarring. Herpes zoster may mimic odontogenic pain during the prodromal stage of the disease. Reactivation of the virus has also been implicated in the pathogenesis of pulpal pathoses. These paradoxical facets are of interest to the endodontist and should be considered in the differential diagnosis of the disease.

Predicting risk for bisphosphonate-related osteonecrosis of the jaws: CTX versus radiographic markers.

BACKGROUND AND OBJECTIVE: The most common risk factor for bisphosphonate-related osteonecrosis of the jaws (BRONJ) is dentoalveolar surgery. It has been suggested that reduced serum C-terminal telopeptide (CTX) can determine the degree of osteoclast suppression and may predict the development of BRONJ after dentoalveolar surgery. Although there are many radiographic appearances associated with BRONJ, there are little data that describes changes preceding dentoalveolar surgery. The objective of this retrospective study was: 1) to investigate if reduced serum CTX values (i.e., <150 pg/mL) were associated with BRONJ after dentoalveolar surgery; and 2) to determine if specific radiographic changes are associated with teeth that develop BRONJ after extraction. STUDY DESIGN: A retrospective review of radiographic and/or serum CTX data was performed for 68 patients with a history of bisphosphonate therapy who either underwent dental extraction or were diagnosed with BRONJ in the Department of Oral and Maxillofacial Surgery during the period 2007-2009. Postoperative healing was assessed for 26 patients with reduced serum CTX levels (<150 pg/mL) who either underwent dental extraction or treatment for BRONJ. Preoperative radiographs were evaluated for 55 patients who either healed normally or developed BRONJ after dental extraction. RESULTS: All 26 patients (100%) who had serum CTX levels <150 pg/mL healed successfully after dentoalveolar surgery (20 patients) or after treatment for BRONJ (6 patients). Among the 55 patients who underwent radiographic evaluation, 24 patients (83%) with BRONJ exhibited periodontal ligament (PDL) widening associated with extracted teeth, whereas only 3 patients (11%) who healed normally demonstrated PDL widening. CONCLUSION: These data suggest that radiographic PDL widening may be a more sensitive indicator than CTX testing in predicting risk of BRONJ. Current guidelines that recommend minimal surgical intervention may need to be revised to include alternative strategies for the elimination or management of this pathology.