Improving antimicrobial release kinetics from hydrophobic polymer implants via ion pairing to combat biomedical-device related infection

Purpose The aim of this study is to improve the drug release properties of antimicrobial agents from hydrophobic biomaterials using using an ion pairing strategy. In so doing antimicrobial agents may be eluted and maintained over a sufficient time period thereby preventing bacterial colonisation and subsequent biofilm formation on medical devices. Methods The model antimicrobial agent was chlorhexidine and the selected fatty acid counter ions were capric acid, myristic acid and stearic acid. The polymethyl methacrylate films were loaded with 2% of fatty acid:antimicrobial agent at the following molar ratios; 0.5:1M, 1:1M and 2:1M and thermally polymerized using azobisisobutyronitrile initiator. Drug release experiments were subsequently performed over a 3-month period and the mass of drug released under sink conditions (pH 7.0, 37oC) quantified using a validated HPLC-UV method. Results In all platforms, a burst of chlorhexidine release was observed over the initial 24-hour period. Similar release kinetics were observed between the formulations during the initial 28 days. However, as time progressed, the chlorhexidine baseline plateaued after 56 days whereas formulations containing the counterions appeared to continuously elute linearly with time. As can be observed in figure 1, the rank order of total chlorhexidine release in the presence of 0.5M fatty acid was myristic acid (40%) > capric acid (35%) > stearic acid (30%)> chlorhexidine baseline (15%). Conclusion The incorporation of fatty acids within the formulation significantly improved chlorhexidine solubility within both the monomer and the polymer and enhanced the drug release kinetics over the period of study. This is attributed to the greater diffusivity of chlorhexidine through PMMA in the presence of fatty acids. In th absence of fatty acids, chlorhexidine release was facilitated by dissolution of surface associated drug particles. This study has illustrated the ability of fatty acids to modulate chlorhexidine release from a model biomaterial through enhanced diffusivity. This strategy may prove advantageous for improved medical devices with enhanced resistance to infection.

WS02.7 Initial and chronic MRSA infection in cystic fibrosis

Objectives Chronic MRSA infection, which affects approximately 26% of CF patients in the USA, is associated with declining lung function and poor outcomes (Dasenbrook, 2010). Anaerobic niches have been described within the CF lung, potentially influencing the virulence of MRSA. This study aims to compare initial and chronic CF MRSA isolates, following aerobic and anaerobic culture. Methods Isolates, obtained from CF sputum at first isolation [“early” (n = 10)] or up to 5 years later, during chronic infection [“late” (n = 15)] were cultured in aerobic and anaerobic conditions. Differences in virulence were compared using the Galleria mellonella infection model. Biofilm formation of each isolate was assessed following staining with crystal violet. Production of Δ-haemolysin (Δ-hly), a surrogate marker for expression of the virulence regulator agr, was determined by haemolysis assay. Results MRSA grown in anaerobic conditions had significantly increased virulence in the G. mellonella model (p = 0.007), increased biofilm formation (p = 0.006) and increased Δ-hly production (p<0.0001). No significant difference between Δ-hly production or biofilm formation were observed between early and late isolates; however late isolates were found to be more virulent in the G. mellonella model (p = 0.0002). Conclusion These results suggest that an anaerobic environment, as found in the CF lung, may increase virulence of MRSA and aid in the establishment of chronic infection. Further clinical studies are required to determine how these phenotypic changes are associated with transition to chronic infection and patient outcome.

The antimicrobial potential of ionic liquids: A source of chemical diversity for infection and biofilm control

Although described almost a century ago, interest in ionic liquids has flourished in the last two decades, with significant advances in the understanding of their chemical, physical and biological property sets driving their widespread application across multiple and diverse research areas. Significant progress has been made through the contributions of numerous research groups detailing novel libraries of ionic liquids, often ‘task-specific’ designer solvents for application in areas as diverse as separation technology, catalysis and bioremediation. Basic antimicrobial screening has often been included as a surrogate indication of the environmental impact of these compounds widely regarded as ‘green’ solvents. Obviating the biological properties, specifically toxicity, of these compounds has obstructed their potential application as sophisticated designer biocides. A recent tangent in ionic liquids research now aims to harness tuneable biological properties of these compounds in the design of novel potent antimicrobials, recognising their unparalleled flexibility for chemical diversity in a severely depleted antimicrobial arsenal. This review concentrates primarily on the antimicrobial potential of ionic liquids and aims to consolidate contemporary microbiological background information, assessment protocols and future considerations necessary to advance the field in light of the urgent need for antimicrobial innovation.

Infection of donor lymphocytes with human T lymphotrophic virus type 1 (HTLV-I) following allogeneic bone marrow transplantation for HTLV-I positive adult T-cell leukaemia

Human T lymphotrophic virus type 1 (HTLV-I) associated leukaemia has a poor prognosis even with chemotherapy. We describe a patient with adult T-cell leukaemia treated with allogeneic bone marrow transplantation from an HTLV-I negative identical sibling donor. During follow-up after bone marrow transplantation, HTLV-I could be repeatedly isolated inspite of anti-viral prophylaxis. The patient died of an acute encephalitis and HTLV-I could be detected in autopsy material from the brain. By a PCR-based technique using short tandem repeats (STRs) it was shown that the patient's haemopoiesis was of donor origin. This shows the infection of donor cells in vivo by an aetiological agent which has been implicated in the leukaemogenic process for adult T-cell leukaemia.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

Respiratory Syncytial Virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanised monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F-protein with subnanomolar affinity. ALX-0171 demonstrated superior in vitro neutralisation compared to palivizumab against prototypic RSV A and B strains. Moreover, ALX-0171 completely blocked replication below limit of detection in 87% of the viruses tested versus 18% for palivizumab at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae

Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.

Antifungal Activity of LL-37 and Truncated Mimetics

Background: Candida albicans is a commensal organism and a constituent of the normal oral flora. Cell concentrations of 1x102 cells/ml and below are indicative of commensal colonisation in the oral cavity, above this level C. albicans can become an opportunistic pathogen; it is the most prevalent human fungal pathogen and a causal agent of the oral infection, candidiasis. The capacity of C. albicans to cause infection arises from its ability to exist in a biofilm ecosystem. Mature C. albicans biofilms display a high level of resistance to antifungals and the need for other therapeutic options has become paramount. Objectives: The objectives of the current study were to determine the antifungal activity of LL-37 (a member of the human cathelicidin family) and two truncated peptide mimetics against C. albicans in both planktonic and biofilm form. Methods: Radial diffusion assays were used to obtain the minimum inhibitory concentration (MIC) of LL-37 and the truncated mimetics KE-18 and KR-12 against planktonic C. albicans. A 96 well microtitre plate assay was employed to study the effects of the peptides on early candida biofilm formation (up to 24 hours) compared with the antifungal drug fluconazole. Biofilm quantification was achieved using the crystal violet assay. Results: MIC values obtained: LL-37 >250µg/ml; KE-18 51µg/ml; and KR-12 11µg/ml. LL-37 significantly reduced the quantity of biofilm formed by C.albicans at both the 4 h and 24 h timepoints (p <0.0001). KE-18 showed significant biofilm reduction over 4 h and 24 h (p=0.0002, p=0.013 respectively), KR-12 showed significant reduction at the 24 h time point only (p=0.0256). Conclusions: Results suggest that LL-37 has the ability to disrupt early biofilm formation of C. albicans with its potency of action similar with that of fluconazole.