Thrombocidin-1-derived antimicrobial peptide TC19 combats superficial multi-drug resistant bacterial wound infections.

Antimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the identification and characterization of the synthetic peptide TC19, derived from the human thrombocidin-1-derived peptide L3. Biophysical experiments into the interaction between TC19 and mimics of human and bacterial plasma membranes demonstrated that the peptide is highly selective for bacterial membranes. In agreement, TC19 combined low cytotoxicity towards human fibroblasts with efficient and rapid killing in human plasma of MDR strains of several bacterial species of the ESKAPE panel. In addition, TC19 induced minor resistance in vitro, neutralized pro-inflammatory activity of bacterial cell envelope components while displaying slight chemotactic activity for human neutrophils. Importantly, topical application of TC19-containing hypromellose gel significantly reduced numbers of viable methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii in a superficial wound infection in mice. Together, TC19 is an attractive candidate for further development as a novel agent against (MDR) bacterial skin wound infections.

The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms.

Development of novel antimicrobial agents is a top priority in the fight against multidrug-resistant (MDR) and persistent bacteria. We developed a panel of synthetic antimicrobial and antibiofilm peptides (SAAPs) with enhanced antimicrobial activities compared to the parent peptide, human antimicrobial peptide LL-37. Our lead peptide SAAP-148 was more efficient in killing bacteria under physiological conditions in vitro than many known preclinical- and clinical-phase antimicrobial peptides. SAAP-148 killed MDR pathogens without inducing resistance, prevented biofilm formation, and eliminated established biofilms and persister cells. A single 4-hour treatment with hypromellose ointment containing SAAP-148 completely eradicated acute and established, biofilm-associated infections with methicillin-resistant Staphylococcus aureus and MDR Acinetobacter baumannii from wounded ex vivo human skin and murine skin in vivo. Together, these data demonstrate that SAAP-148 is a promising drug candidate in the battle against antibiotic-resistant bacteria that pose a great threat to human health.

Silver-coated endotracheal tubes for prevention of ventilator-associated pneumonia in critically ill patients.

BACKGROUND: Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections in intubated and mechanically ventilated patients. Endotracheal tubes (ETTs) appear to be an independent risk factor for VAP. Silver-coated ETTs slowly release silver cations. It is these silver ions that appear to have a strong antimicrobial effect. Because of this antimicrobial effect of silver, silver-coated ETTs could be an effective intervention to prevent VAP in people who require mechanical ventilation for 24 hours or longer. OBJECTIVES: Our primary objective was to investigate whether silver-coated ETTs are effective in reducing the risk of VAP and hospital mortality in comparison with standard non-coated ETTs in people who require mechanical ventilation for 24 hours or longer. Our secondary objective was to ascertain whether silver-coated ETTs are effective in reducing the following clinical outcomes: device-related adverse events, duration of intubation, length of hospital and intensive care unit (ICU) stay, costs, and time to VAP onset. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2014 Issue 10, MEDLINE, EMBASE, EBSCO CINAHL, and reference lists of trials. We contacted corresponding authors for additional information and unpublished studies. We did not impose any restrictions on the basis of date of publication or language. The date of the last search was October 2014. SELECTION CRITERIA: We included all randomized controlled trials (RCTs) and quasi-randomized trials that evaluated the effects of silver-coated ETTs or a combination of silver with any antimicrobial-coated ETTs with standard non-coated ETTs or with other antimicrobial-coated ETTs in critically ill people who required mechanical ventilation for 24 hours or longer. We also included studies that evaluated the cost-effectiveness of silver-coated ETTs or a combination of silver with any antimicrobial-coated ETTs. DATA COLLECTION AND ANALYSIS: Two review authors (GT, HV) independently extracted the data and summarized study details from all included studies using the specially designed data extraction form. We used standard methodological procedures expected by The Cochrane Collaboration. We performed meta-analysis for outcomes when possible. MAIN RESULTS: We found three eligible randomized controlled trials, with a total of 2081 participants. One of the three included studies did not mention the amount of participants and presented no outcome data. The 'Risk of bias' assessment indicated that there was a high risk of detection bias owing to lack of blinding of outcomes assessors, but we assessed all other domains to be at low risk of bias. Trial design and conduct were generally adequate, with the most common areas of weakness in blinding. The majority of participants were included in centres across North America. The mean age of participants ranged from 61 to 64 years, and the mean duration of intubation was between 3.2 and 7.7 days. One trial comparing silver-coated ETTs versus non-coated ETTs showed a statistically significant decrease in VAP in favour of the silver-coated ETT (1 RCT, 1509 participants; 4.8% versus 7.5%, risk ratio (RR) 0.64, 95% confidence interval (CI) 0.43 to 0.96; number needed to treat for an additional beneficial outcome (NNTB) = 37; low-quality evidence). The risk of VAP within 10 days of intubation was significantly lower with the silver-coated ETTs compared with non-coated ETTs (1 RCT, 1509 participants; 3.5% versus 6.7%, RR 0.51, 95% CI 0.31 to 0.82; NNTB = 32; low-quality evidence). Silver-coated ETT was associated with delayed time to VAP occurrence compared with non-coated ETT (1 RCT, 1509 participants; hazard ratio 0.55, 95% CI 0.37 to 0.84). The confidence intervals for the results of the following outcomes did not exclude potentially important differences with either treatment. There were no statistically significant differences between groups in hospital mortality (1 RCT, 1509 participants; 30.4% versus 26.6%, RR 1.09, 95% CI 0.93 to 1.29; low-quality evidence); device-related adverse events (2 RCTs, 2081 participants; RR 0.65, 95% CI 0.37 to 1.16; low-quality evidence); duration of intubation; and length of hospital and ICU stay. We found no clinical studies evaluating the cost-effectiveness of silver-coated ETTs. AUTHORS' CONCLUSIONS: This review provides limited evidence that silver-coated ETT reduces the risk of VAP, especially during the first 10 days of mechanical ventilation.

A doxycycline-loaded polymer-lipid encapsulation matrix coating for the prevention of implant-related osteomyelitis due to doxycycline-resistant methicillin-resistant Staphylococcus aureus.

Implant-associated bone infections caused by antibiotic-resistant pathogens pose significant clinical challenges to treating physicians. Prophylactic strategies that act against resistant organisms, such as methicillin-resistant Staphylococcus aureus (MRSA), are urgently required. In the present study, we investigated the efficacy of a biodegradable Polymer-Lipid Encapsulation MatriX (PLEX) loaded with the antibiotic doxycycline as a local prophylactic strategy against implant-associated osteomyelitis. Activity was tested against both a doxycycline-susceptible (doxy(S)) methicillin-susceptible S. aureus (MSSA) as well as a doxycycline-resistant (doxy(R)) methicillin-resistant S. aureus (MRSA). In vitro elution studies revealed that 25% of the doxycycline was released from the PLEX-coated implants within the first day, followed by a 3% release per day up to day 28. The released doxycycline was highly effective against doxy(S) MSSA for at least 14days in vitro. A bolus injection of doxycycline mimicking a one day release from the PLEX-coating reduced, but did not eliminate, mouse subcutaneous implant-associated infection (doxy(S) MSSA). In a rabbit intramedullary nail-related infection model, all rabbits receiving a PLEX-doxycycline-coated nail were culture negative in the doxy(S) MSSA-group and the surrounding bone displayed a normal physiological appearance in both histological sections and radiographs. In the doxy(R) MRSA inoculated rabbits, a statistically significant reduction in the number of culture-positive samples was observed for the PLEX-doxycycline-coated group when compared to the animals that had received an uncoated nail, although the reduction in bacterial burden did not reach statistical significance. In conclusion, the PLEX-doxycycline coating on titanium alloy implants provided complete protection against implant-associated MSSA osteomyelitis, and resulted in a significant reduction in the number of culture positive samples when challenged with a doxycycline-resistant MRSA.

Staphylococcus epidermidis originating from titanium implants infects surrounding tissue and immune cells.

Infection is a major cause of failure of inserted or implanted biomedical devices (biomaterials). During surgery, bacteria may adhere to the implant, initiating biofilm formation. Bacteria are also observed in and recultured from the tissue surrounding implants, and may even reside inside host cells. Whether these bacteria originate from biofilms is not known. Therefore, we investigated the fate of Staphylococcus epidermidis inoculated on the surface of implants as adherent planktonic cells or as a biofilm in mouse experimental biomaterial-associated infection. In order to discriminate the challenge strain from potential contaminating mouse microflora, we constructed a fully virulent green fluorescent S. epidermidis strain. S. epidermidis injected along subcutaneous titanium implants, pre-seeded on the implants or pre-grown as biofilm, were retrieved from the implants as well as the surrounding tissue in all cases after 4days, and in histology bacteria were observed in the tissue co-localizing with macrophages. Thus, bacteria adherent to or in a biofilm on the implant are a potential source of infection of the surrounding tissue, and antimicrobial strategies should prevent both biofilm formation and tissue colonization.

Antibacterial components of honey.

The antibacterial activity of honey has been known since the 19th century. Recently, the potent activity of honey against antibiotic-resistant bacteria has further increased the interest for application of honey, but incomplete knowledge of the antibacterial activity is a major obstacle for clinical applicability. The high sugar concentration, hydrogen peroxide, and the low pH are well-known antibacterial factors in honey and more recently, methylglyoxal and the antimicrobial peptide bee defensin-1 were identified as important antibacterial compounds in honey. The antibacterial activity of honey is highly complex due to the involvement of multiple compounds and due to the large variation in the concentrations of these compounds among honeys. The current review will elaborate on the antibacterial compounds in honey. We discuss the activity of the individual compounds, their contribution to the complex antibacterial activity of honey, a novel approach to identify additional honey antibacterial compounds, and the implications of the novel developments for standardization of honey for medical applications.

Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

Two major medicinal honeys have different mechanisms of bactericidal activity.

Honey is increasingly valued for its antibacterial activity, but knowledge regarding the mechanism of action is still incomplete. We assessed the bactericidal activity and mechanism of action of Revamil® source (RS) honey and manuka honey, the sources of two major medical-grade honeys. RS honey killed Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within 2 hours, whereas manuka honey had such rapid activity only against B. subtilis. After 24 hours of incubation, both honeys killed all tested bacteria, including methicillin-resistant Staphylococcus aureus, but manuka honey retained activity up to higher dilutions than RS honey. Bee defensin-1 and H2O2 were the major factors involved in rapid bactericidal activity of RS honey. These factors were absent in manuka honey, but this honey contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a major bactericidal factor in manuka honey, but after neutralization of this compound manuka honey retained bactericidal activity due to several unknown factors. RS and manuka honey have highly distinct compositions of bactericidal factors, resulting in large differences in bactericidal activity.

Exploring platelet chemokine antimicrobial activity: nuclear magnetic resonance backbone dynamics of NAP-2 and TC-1.

The platelet chemokines neutrophil-activating peptide-2 (NAP-2) and thrombocidin-1 (TC-1) differ by only two amino acids at their carboxy-terminal ends. Nevertheless, they display a significant difference in their direct antimicrobial activities, with the longer NAP-2 being inactive and TC-1 being active. In an attempt to rationalize this difference in activity, we studied the structure and the dynamics of both proteins by nuclear magnetic resonance (NMR) spectroscopy. Using 15N isotope-labeled protein, we confirmed that the two monomeric proteins essentially have the same overall structure in aqueous solution. However, NMR relaxation measurements provided evidence that the negatively charged carboxy-terminal residues of NAP-2 experience a restricted motion, whereas the carboxy-terminal end of TC-1 moves in an unrestricted manner. The same behavior was also seen in molecular dynamic simulations of both proteins. Detailed analysis of the protein motions through model-free analysis, as well as a determination of their overall correlation times, provided evidence for the existence of a monomer-dimer equilibrium in solution, which seemed to be more prevalent for TC-1. This finding was supported by diffusion NMR experiments. Dimerization generates a larger cationic surface area that would increase the antimicrobial activities of these chemokines. Moreover, these data also show that the negatively charged carboxy-terminal end of NAP-2 (which is absent in TC-1) folds back over part of the positively charged helical region of the protein and, in doing so, interferes with the direct antimicrobial activity.

How honey kills bacteria.

With the rise in prevalence of antibiotic-resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria tested, including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producing Escherichia coli, ciprofloxacin-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus faecium, were killed by 10-20% (v/v) honey, whereas > or = 40% (v/v) of a honey-equivalent sugar solution was required for similar activity. Honey accumulated up to 5.62 +/- 0.54 mM H(2)O(2) and contained 0.25 +/- 0.01 mM methylglyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity. Using B. subtilis for activity-guided isolation of the additional antimicrobial factors, we discovered bee defensin-1 in honey. After combined neutralization of H(2)O(2), MGO, and bee defensin-1, 20% honey had only minimal activity left, and subsequent adjustment of the pH of this honey from 3.3 to 7.0 reduced the activity to that of sugar alone. Activity against all other bacteria tested depended on sugar, H(2)O(2), MGO, and bee defensin-1. Thus, we fully characterized the antibacterial activity of medical-grade honey.

Medical-grade honey kills antibiotic-resistant bacteria in vitro and eradicates skin colonization.

BACKGROUND: Antibiotic resistance among microbes urgently necessitates the development of novel antimicrobial agents. Since ancient times, honey has been used successfully for treatment of infected wounds, because of its antibacterial activity. However, large variations in the in vitro antibacterial activity of various honeys have been reported and hamper its acceptance in modern medicine. METHODS: We assessed the in vitro bactericidal activity of Revamil (Bfactory), a medical-grade honey produced under controlled conditions, and assessed its efficacy for reduction of forearm skin colonization in healthy volunteers in a within-subject-controlled trial. RESULTS: With Bacillus subtilis as a test strain, we demonstrated that the variation in bactericidal activity of 11 batches of medical-grade honey was <2-fold. Antibiotic-susceptible and -resistant isolates of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella oxytoca were killed within 24 h by 10%-40% (vol/vol) honey. After 2 days of application of honey, the extent of forearm skin colonization in healthy volunteers was reduced 100-fold (P < .001), and the numbers of positive skin cultures were reduced by 76% (P < .001). CONCLUSIONS: Revamil is a promising topical antimicrobial agent for prevention or treatment of infections, including those caused by multidrug-resistant bacteria.

Treatment and prevention of Staphylococcus epidermidis experimental biomaterial-associated infection by bactericidal peptide 2.

Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices and are predominantly caused by staphylococci, especially Staphylococcus epidermidis. We investigated the in vitro microbicidal activity of the synthetic antimicrobial peptide bactericidal peptide 2 (BP2) and its efficacy in a murine model of S. epidermidis BAI. BP2 showed potent microbicidal activity at micromolar concentrations against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The staphylocidal activity of BP2 was not affected by physiological salt concentrations and was only slightly affected by the presence of human plasma. In the BAI model, injection of BP2 (5 mg/kg of body weight) 1 h after challenge with S. epidermidis resulted in an 80% reduction in the number of culture-positive implants and a 100-fold reduction in survival of S. epidermidis in peri-implant tissue at 24 h postchallenge. When BP2 was injected along implants 3 h prior to bacterial challenge, the median numbers of CFU cultured from biomaterial implants and peri-implant tissue were reduced by 85% and 90%, respectively. In conclusion, BP2 has potent, broad-spectrum in vitro microbicidal activity and showed potent in vivo activity in a murine model of S. epidermidis biomaterial-associated infection.