Journal Article

Animals; Bioware; Bone Neoplasms; Collagen; Dermatitis; Disease Models, Animal; Drug Carriers; Gene Therapy; Humans; Hypersensitivity; Mice; Mice, Nude; Nanoparticles; Neoplasm Metastasis; Oligonucleotides; PC-3M-luc; RNA, Small Interfering; Tissue Distribution

The goal of our research is to provide a practical platform for drug delivery in oligonucleotide therapy. We report here the efficacy of an atelocollagen-mediated oligonucleotide delivery system applied to systemic siRNA and antisense oligonucleotide treatments in animal disease models. Atelocollagen and oligonucleotides formed a complex of nanosized particles, which was highly stable against nucleases. The complex allowed oligonucleotides to be delivered efficiently into several organs and tissues via intravenous administration. In a tumor metastasis model, the complex successfully delivered siRNA to metastasized tumors in bone tissue and inhibited their growth. We also demonstrated that a single intravenous treatment of the antisense oligodeoxynucleotide complex suppressed ear dermatitis in a contact hypersensitivity model. These results indicate the strong potential of the atelocollagen-mediated drug delivery system for practical therapeutic technology.

Journal Article

Aza Compounds/pharmacology, Biofilms/drug effects/*growth & development, Electricity/*adverse effects, Pseudomonas/drug effects/*growth & development, Quinolines/pharmacology, Staphylococcus/drug effects/*growth & development, Tobramycin/pharmacology IVIS, Xenogen, Xen30

The activity of electrical current against planktonic bacteria has previously been demonstrated. The short-term exposure of the bacteria in biofilms to electrical current in the absence of antimicrobials has been shown to have no substantial effect; however, longer-term exposure has not been studied. A previously described in vitro model was used to determine the effect of prolonged exposure (i.e., up to 7 days) to low-intensity (i.e., 20-, 200-, and 2,000-microampere) electrical direct currents on Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis biofilms. Dose- and time-dependent killing was observed. A maximum of a 6-log(10)-CFU/cm(2) reduction was observed when S. epidermidis biofilms were exposed to 2,000 microamperes for at least 2 days. A 4- to 5-log(10)-CFU/cm(2) reduction was observed when S. aureus biofilms were exposed to 2,000 microamperes for at least 2 days. Finally, a 3.5- to 5-log(10)-CFU/cm(2) reduction was observed when P. aeruginosa biofilms were exposed to electrical current for 7 days. A higher electrical current intensity correlated with greater decreases in viable bacteria at all time points studied. In conclusion, low-intensity electrical current substantially reduced the numbers of viable bacteria in staphylococcal or Pseudomonas biofilms, a phenomenon we have labeled the “electricidal effect.”

Journal Article

Xen31, Xen 31, MRSA, S. aureus, IVIS, Bioluminescence

Biofilms that develop on indwelling devices are a major concern in clinical settings. While removal of colonized devices remains the most frequent strategy for avoiding device-related complications, antibiotic lock therapy constitutes an adjunct therapy for catheter-related infection. However, currently used antibiotic lock solutions are not fully effective against biofilms, thus warranting a search for new antibiotic locks. Metal-binding chelators have emerged as potential adjuvants due to their dual anticoagulant/antibiofilm activities, but studies investigating their efficiency were mainly in vitro or else focused on their effects in prevention of infection. To assess the ability of such chelators to eradicate mature biofilms, we used an in vivo model of a totally implantable venous access port inserted in rats and colonized by either Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, or Pseudomonas aeruginosa. We demonstrate that use of tetrasodium EDTA (30 mg/ml) as a supplement to the gentamicin (5 mg/ml) antibiotic lock solution associated with systemic antibiotics completely eradicated Gram-positive and Gram-negative bacterial biofilms developed in totally implantable venous access ports. Gentamicin-EDTA lock was able to eliminate biofilms with a single instillation, thus reducing length of treatment. Moreover, we show that this combination was effective for immunosuppressed rats. Lastly, we demonstrate that a gentamicin-EDTA lock is able to eradicate the biofilm formed by a gentamicin-resistant strain of methicillin-resistant S. aureus. This in vivo study demonstrates the potential of EDTA as an efficient antibiotic adjuvant to eradicate catheter-associated biofilms of major bacterial pathogens and thus provides a promising new lock solution.

Journal Article

Animals, Anti-Bacterial Agents/ pharmacology, Bacterial Infections/drug therapy/microbiology, Biofilms/ drug effects/growth & development, Bioware; Catheterization/adverse effects, Chemiluminescent Measurements, Ciprofloxacin/pharmacology, Colony Count, Microbial, Disease Models, Animal, Dose-Response Relationship, Drug, Drug Monitoring/methods, Mice, Rifampin/pharmacology, Staphylococcus aureus/drug effects/genetics/growth & development, Tobramycin/pharmacology IVIS, Xenogen; Xen29

We have developed a rapid, continuous method for monitoring the effectiveness of several antibacterial agents in real time, noninvasively, by using a recently described mouse model of chronic biofilm infection (J. L. Kadurugamuwa et al., Infect. Immun. 71:882-890, 2003), which relies on biophotonic imaging of bioluminescent bacteria. To facilitate real-time monitoring of infection, we used a Staphylococcus aureus isolate that was made bioluminescent by inserting a modified lux operon into the bacterial chromosome. This bioluminescent reporter bacterium was used to study the antimicrobial effects of several antibiotics belonging to different molecular families. Treatment with rifampin, tobramycin, and ciprofloxacin was started 7 days after subcutaneous implantation of catheters precolonized with 10(4) CFU of S. aureus. Three different doses of antibiotics were administered twice a day for 4 consecutive days. The number of metabolically active bacteria in untreated mice and the tobramycin- and ciprofloxacin-treated groups remained relatively unchanged over the 4-week observation period, indicating poor efficacies for tobramycin and ciprofloxacin. A rapid dose-dependent decline in metabolic activity in rifampin-treated groups was observed, with almost a 90% reduction after two doses and nearly undetectable levels after three doses. The disappearance of light emission correlated with colony counts. After the final treatment, cell numbers rebounded as a function of concentration in a time-dependent manner. The staphylococci isolated from the catheters of mice treated with rifampin were uniformly resistant to rifampin but retained their in vitro susceptibilities to tobramycin and ciprofloxacin. Since the metabolic activities of viable cells and a postantibiotic effect could be detected directly on the support matrix nondestructively and noninvasively, the methodology is specifically appealing for investigating the effects of antibiotics on biofilms in vivo. Moreover, our study points to the possible use of biophotonic imaging for the detection of the development of resistance to therapeutic agents during treatment of chronic infections in vivo.

Journal Article

Acetamides; Animals; Anti-Bacterial Agents; Bioware; Colony Count, Microbial; Daptomycin; Female; Luminescent Measurements; Methicillin Resistance; Mice; Microbial Sensitivity Tests; Neutropenia; Oxazolidinones; Peritonitis; Staphylococcus aureus; Xen29

The rising rates of antibiotic resistance accentuate the critical need for new antibiotics. Daptomycin is a new antibiotic with a unique mode of action and a rapid in vitro bactericidal effect against gram-positive organisms. This study examined the kinetics of daptomycin's bactericidal action against peritonitis caused by methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in healthy and neutropenic mice and compared this activity with those of other commonly used antibiotics. CD-1 mice were inoculated intraperitoneally with lethal doses of MSSA (Xen-29) or MRSA (Xen-1), laboratory strains transformed with a plasmid containing the lux operon, which confers bioluminescence. One hour later, the animals were given a single dose of daptomycin at 50 mg/kg of body weight subcutaneously (s.c.), nafcillin at 100 mg/kg s.c., vancomycin at 100 mg/kg s.c., linezolid at 100 mg/kg via gavage (orally), or saline (10 ml/kg s.c.). The mice were anesthetized hourly, and photon emissions from living bioluminescent bacteria were imaged and quantified. The luminescence in saline-treated control mice either increased (neutropenic mice) or remained relatively unchanged (healthy mice). In contrast, by 2 to 3 h postdosing, daptomycin effected a 90% reduction of luminescence of MSSA or MRSA in both healthy and neutropenic mice. The activity of daptomycin against both MSSA and MRSA strains was superior to those of nafcillin, vancomycin, and linezolid. Against MSSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than nafcillin or linezolid. Against MRSA peritonitis, daptomycin showed greater and more rapid bactericidal activity than vancomycin or linezolid. The rapid decrease in the luminescent signal in the daptomycin-treated neutropenic mice underscores the potency of this antibiotic against S. aureus in the immune-suppressed host.