An Australian native plant: A novel antimicrobial for the treatment of MRSA

Chronic wounds, often associated with venous and arterial ulcers, diabetes and pressure sores, is an area of great concern. In Australia, the cost of treating chronic wounds is conservatively estimated at $285 million/annum for the treatment of pressure ulcers and $654 million annually for the treatment and management of leg ulcers. Current figures indicate that more than seven million people suffer from chronic wounds worldwide with Australians accounting for approximately 600,000 of this number. Bacterial infection of the wound site is a major issue as contamination of a chronic wound with methicillin-resistant Staphylococcus aureus (MRSA) significantly delays wound healing. Further, once systemic, current antibiotic therapies capable of treating the infection are limited. Aboriginal bush medicine has been used for thousands of years for the treatment of wounds and sores. Hence, we selected a native Australian plant to evaluate its bactericidal activity against MRSA.

The ADMIN-ICU survey: A survey on antimicrobial dosing and monitoring in ICUs

Objectives: There is little evidence and few guidelines to inform the most appropriate dosing and monitoring for antimicrobials in the ICU. We aimed to survey current practices around the world. Methods: An online structured questionnaire was developed and sent by e-mail to obtain information on local antimicrobial prescribing practices for glycopeptides, piperacillin/tazobactam, carbapenems, aminoglycosides and colistin. Results: A total of 402 professionals from 328 hospitals in 53 countries responded, of whom 78% were specialists in intensive care medicine (41% intensive care, 30% anaesthesiology, 14% internal medicine) and 12% were pharmacists. Vancomycin was used as a continuous infusion in 31% of units at a median (IQR) daily dose of 25 (25–30) mg/kg. Piperacillin/tazobactam was used as an extended infusion by 22% and as a continuous infusion by 7%. An extended infusion of carbapenem (meropenem or imipenem) was used by 27% and a continuous infusion by 5%. Colistin was used at a daily dose of 7.5 (3.9–9) million IU (MIU)/day, predominantly as a short infusion. The most commonly used aminoglycosides were gentamicin (55%) followed by amikacin (40%), with administration as a single daily dose reported in 94% of the cases. Gentamicin was used at a daily dose of 5 (5–6) mg/day and amikacin at a daily dose of 15 (15–20) mg/day. Therapeutic drug monitoring of vancomycin, piperacillin/tazobactam and meropenem was used by 74%, 1% and 2% of the respondents, respectively. Peak aminoglycoside concentrations were sampled daily by 28% and trough concentrations in all patients by 61% of the respondents. Conclusions: We found wide variability in reported practices for antibiotic dosing and monitoring. Research is required to develop evidence-based guidelines to standardize practices.

Unique and cross-reactive T cell epitope peptides of the major bahia grass pollen allergen, pas n 1

Background Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Antimicrobial treatment of non-cystic fibrosis bronchiectasis

Bronchiectasis unrelated to cystic fibrosis is characterized by chronic wet or productive cough, recurrent exacerbations and irreversible bronchial dilatation. After antibiotics and vaccines became available and living standards in affluent countries improved, its resulting reduced prevalence meant bronchiectasis was considered an ‘orphan disease’. This perception has changed recently with increasing use of CT scans to diagnose bronchiectasis, including in those with severe chronic obstructive pulmonary disease or ‘difficult to control’ asthma, and adds to its already known importance in non-affluent countries and disadvantaged Indigenous communities. Following years of neglect, there is renewed interest in identifying the pathogenetic mechanisms of bronchiectasis, including the role of infection, and conducting clinical trials. This is providing much needed evidence to guide antimicrobial therapy, which has relied previously upon extrapolating treatments used in cystic fibrosis and chronic obstructive pulmonary disease. While many knowledge gaps and management challenges remain, the future is improving for patients with bronchiectasis.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

A study of a retention of antimicrobial activity by plasma polymerized terpinen-4-ol thin films

Terpinen-4-ol is the main constituent of Melaleuca alternifolia essential oil known for its biocidal and anti-inflammatory properties. The possibility of fabricating polymer thin films from terpinen-4-ol using radio frequency (RF) plasma polymerisation for the prevention of the growth of Pseudomonas aeruginosa was investigated, and the properties of the resultant films compared against their biologically active precursor. Films fabricated at 10 W prevented bacterial attachment and EPS secretion, whilst polyterpenol films deposited at 25 W demonstrated no biocidal activity against the pathogen.

Cytotoxic Effects and Biocompatibility of Antimicrobial Materials

The rising demand for medical implants for ageing populations and ongoing advancements in medical technology continue to drive the use of implantable devices. Higher implant usage has a consequent increased incidence of implant-related infections, and associated prolonged patient care, pain and loss of limb and other organ function. Numerous antibacterial surfaces have been designed that prevent the onset of biofilm formation, thus reducing or preventing implant-associated infections through inhibiting bacterial adhesion or by killing the organisms that successfully attach to the surface of the implant. Other surfaces have been designed to stimulate a local immune response, promoting the natural clearing of the invading pathogen. The desired antibacterial effects are typically achieved by modulating the surface chemistry and morphology of the implant material, by means of the controlled release of pharmacological agents and bioactive compounds from the surface of the material, or by a combination of both processes. An important issue for any type of antibacterial surface modification lies in balancing the non-fouling, bacteriostatic or bactericidal effects against local and systemic biocompatibility. In this chapter, we will first describe the concept of biocompatibility and its evolution, from devices that do not evoke a negative host response to those that actively drive host regeneration. We will then review the challenges associated with merging the need for an implant material to withstand a bacterial load with those associated with supporting function restoration and tissue healing.

The need for cost-effectiveness analyses of antimicrobial stewardship programmes: A structured review

The cost effectiveness of antimicrobial stewardship (AMS) programmes was reviewed in hospital settings of Organisation for Economic Co-operation and Development (OECD) countries, and limited to adult patient populations. In each of the 36 studies, the type of AMS strategy and the clinical and cost outcomes were evaluated. The main AMS strategy implemented was prospective audit with intervention and feedback (PAIF), followed by the use of rapid technology, including rapid polymerase chain reaction (PCR)-based methods and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) technology, for the treatment of bloodstream infections. All but one of the 36 studies reported that AMS resulted in a reduction in pharmacy expenditure. Among 27 studies measuring changes to health outcomes, either no change was reported post-AMS, or the additional benefits achieved from these outcomes were not quantified. Only two studies performed a full economic evaluation: one on a PAIF-based AMS intervention; and the other on use of rapid technology for the selection of appropriate treatment for serious Staphylococcus aureus infections. Both studies found the interventions to be cost effective. AMS programmes achieved a reduction in pharmacy expenditure, but there was a lack of consistency in the reported cost outcomes making it difficult to compare between interventions. A failure to capture complete costs in terms of resource use makes it difficult to determine the true cost of these interventions. There is an urgent need for full economic evaluations that compare relative changes both in clinical and cost outcomes to enable identification of the most cost-effective AMS strategies in hospitals.

Antimicrobial efficacy of a novel silver hydrogel dressing compared to two common silver burn wound dressings: Acticoat™ and PolyMem Silver

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles. This study compared the antimicrobial efficacy of this novel dressing to two commercially available silver dressings; Acticoat™ and PolyMem Silver(®). Three different antimicrobial tests were used: disc diffusion, broth culture, and the Live/Dead(®) Baclight™ bacterial viability assay. Burn wound pathogens (P. aeruginosa, MSSA, A. baumannii and C. albicans) and antibiotic resistant strains (MRSA and VRE) were tested. All three antimicrobial tests indicated that Acticoat™ was the most effective antimicrobial agent, with inhibition zone lengths of 13.9-18.4mm. It reduced the microbial inocula below the limit of detection (10(2)CFU/ml) and reduced viability by 99% within 4h. PolyMem Silver(®) had no zone of inhibition for most tested micro-organisms, and it also showed poor antimicrobial activity in the broth culture and Live/Dead(®) Baclight™ assays. Alarmingly, it appeared to promote the growth of VRE. The silver hydrogel reduced most of the tested microbial inocula below the detection limit and decreased bacterial viability by 94-99% after 24h exposure. These results support the possibility of using this novel silver hydrogel as a burn wound dressing in the future

Computational methods for the prediction of the structure and interactions of coiled-coil peptides

The past several years have seen significant advances in the development of computational methods for the prediction of the structure and interactions of coiled-coil peptides. These methods are generally based on pairwise correlations of amino acids, helical propensity, thermal melts and the energetics of sidechain interactions, as well as statistical patterns based on Hidden Markov Model (HMM) and Support Vector Machine (SVM) techniques. These methods are complemented by a number of public databases that contain sequences, motifs, domains and other details of coiled-coil structures identified by various algorithms. Some of these computational methods have been developed to make predictions of coiled-coil structure on the basis of sequence information; however, structural predictions of the oligomerisation state of these peptides still remains largely an open question due to the dynamic behaviour of these molecules. This review focuses on existing in silico methods for the prediction of coiled-coil peptides of functional importance using sequence and/or three-dimensional structural data.

Free energy calculations of the interactions of c-Jun-based synthetic peptides with the c-Fos protein

The c-Fos–c-Jun complex forms the activator protein 1 transcription factor, a therapeutic target in the treatment of cancer. Various synthetic peptides have been designed to try to selectively disrupt the interaction between c-Fos and c-Jun at its leucine zipper domain. To evaluate the binding affinity between these synthetic peptides and c-Fos, polarizable and nonpolarizable molecular dynamics (MD) simulations were conducted, and the resulting conformations were analyzed using the molecular mechanics generalized Born surface area (MM/GBSA) method to compute free energies of binding. In contrast to empirical and semiempirical approaches, the estimation of free energies of binding using a combination of MD simulations and the MM/GBSA approach takes into account dynamical properties such as conformational changes, as well as solvation effects and hydrophobic and hydrophilic interactions. The predicted binding affinities of the series of c-Jun-based peptides targeting the c-Fos peptide show good correlation with experimental melting temperatures. This provides the basis for the rational design of peptides based on internal, van der Waals, and electrostatic interactions.

General Practitioner Antimicrobial Stewardship Programme Study (GAPS): protocol for a cluster randomised controlled trial

Background There is a strong link between antibiotic consumption and the rate of antibiotic resistance. In Australia, the vast majority of antibiotics are prescribed by general practitioners, and the most common indication is for acute respiratory infections. The aim of this study is to assess if implementing a package of integrated, multifaceted interventions reduces antibiotic prescribing for acute respiratory infections in general practice. Methods/design This is a cluster randomised trial comparing two parallel groups of general practitioners in 28 urban general practices in Queensland, Australia: 14 intervention and 14 control practices. The protocol was peer-reviewed by content experts who were nominated by the funding organization. This study evaluates an integrated, multifaceted evidence-based package of interventions implemented over a six month period. The included interventions, which have previously been demonstrated to be effective at reducing antibiotic prescribing for acute respiratory infections, are: delayed prescribing; patient decision aids; communication training; commitment to a practice prescribing policy for antibiotics; patient information leaflet; and near patient testing with C-reactive protein. In addition, two sub-studies are nested in the main study: (1) point prevalence estimation carriage of bacterial upper respiratory pathogens in practice staff and asymptomatic patients; (2) feasibility of direct measures of antibiotic resistance by nose/throat swabbing. The main outcome data are from Australia’s national health insurance scheme, Medicare, which will be accessed after the completion of the intervention phase. They include the number of antibiotic prescriptions and the number of patient visits per general practitioner for periods before and during the intervention. The incidence of antibiotic prescriptions will be modelled using the numbers of patients as the denominator and seasonal and other factors as explanatory variables. Results will compare the change in prescription rates before and during the intervention in the two groups of practices. Semi-structured interviews will be conducted with the general practitioners and practice staff (practice nurse and/or practice manager) from the intervention practices on conclusion of the intervention phase to assess the feasibility and uptake of the interventions. An economic evaluation will be conducted to estimate the costs of implementing the package, and its cost-effectiveness in terms of cost per unit reduction in prescribing. Discussion The results on the effectiveness, cost-effectiveness, acceptability and feasibility of this package of interventions will inform the policy for any national implementation.

Antimicrobial effects of ethanolic extracts from Cuscuta reflexa Roxb. (Convolvulaceae).

Ethanolic whole plant extracts obtained from Cuscuta reflexa Roxb were screened against Gram positive (Bacillus subtilis and Staphylococcus aureus) and Gram negative (Escherichia coli and Salmonella typhi) bacteria to evaluate their antimicrobial activity. Of the four concentrations of plant extract tested (200 µg/mL, 300 µg/mL, 400 µg/mL or 500 µg/mL), 500 µg/mL elicited the greatest zones of bacterial inhibition across three of the bacteria. In contrast, the growth of Salmonella typhi was not halted regardless of extract concentration. At 200 µg/mL, only the growth of E. coli was inhibited. Overall, although the greatest antimicrobial activity was demonstrated to be against E. coli at a concentration of 500 µg/mL (24.6±0.24), upon comparison to the other bacteria, both B. cereus and S. aureus educed similar zones of inhibition upon comparison to their positive antibiotic control.