Spray dried combinations of lactoferrin with antibiotics appear superior to monotherapy for reducing biofilm formation by pseudomonas aeruginosa

SD Apo Lactoferrin-Tobramycin/Gentamicin Combinations are superior to monotherapy in the eradication of Pseudomonas aeruginosa Biofilm in the lungs Wilson Oguejiofor1, Lindsay J. Marshall1, Andrew J. Ingham1, Robert Price2, Jag. Shur2 1School of Life and Health Sciences, Aston University, Birmingham, UK. 2School of Pharmacy and Pharmacology, University of Bath, Bath, UK. KEYWORDS: lactoferrin, apo lactoferrin, spray drying, biofilm, cystic fibrosis Introduction Chronic lung infections from the opportunistic pathogeen Pseudomonas aeruginosa has been recognised as a major contributor to the incidences of high morbidity and mortality amongst cystic fibrosis (CF) patients (1,2). Currently, strategies for managing lung infections in CF patients involves the aggressive use of aerosolised antibiotics (3), however, increasing evidence suggests that the biofilm component of P. aeruginosa in the lower airway remains unperturbed and is associated with the development of antibiotic resistance. If this is so then, there is an urgent need to suitably adjust the current treatment strategy so that it includes compounds that prevent biofilm formation or disrupt established biofilms. It is well understood that biofilm formation is strongly dependent on iron (Fe3+) availability (4), therefore aerosolised anti-infective formulations which has the ability to chelate iron may essentially be a well suited therapy for eliminating P. aeruginosa biofilms on CF airway epithelial cells (5). In this study, we report the use of combination therapy; an aminoglycosides (tobramycin and gentamicin) and an antimicrobial peptide (lactoferrin) to significantly deplete P. aeruginosa biofilms. We demonstrate that lactoferrin-tobramycin and lactoferrin-gentamicin combinations are superior to the single antibiotic regime currently being employed to combat P. aeruginosa biofilms. MATERIALS AND METHOD Antibiotics: The antibiotics used in this study included gentamicin and tobramycin supplied by Fagron, UK. Bacterial strain and growth conditions: Pseudomonas aeruginosa strain PAO1 was provided by Prof. Peter Lambert of Aston University, Birmingham UK. The Strains were routinely grown from storage in a medium supplemented with magnesium chloride, glucose and casamino acids. Dialysis of lactoferrin: Apo lactoferrin was prepared by dialyzing a suspension of lactoferrin for 24 hrs at 4 °C against 20 mmol/L sodium dihydrogen phosphate, 20 mmol/L sodium acetate and 40 mmol/L EDTA (pH 3.5). Ferric ion (Fe3+) removal was verified by atomic absorption spectroscopy measurements. Spray drying of combinations of lactoferrin and apo lactoferrin with the different aminoglycosides: Combinations of tobramycin and gentamicin with the different preparations of lactoferrin were spray dried (SD) as a 2% (w/v) aqueous suspension. The spray drying parameters utilized for the production of suitable micron-sized particles includes: Inlet temperature, 180°C, spray flow rate, 606 L/hr; pump setting, 10%; aspirator setting, 85% (34m3/hr) to produce various outlet temperatures ranging from 99 - 106°C. Viability assay: To test the bactericidal activity of the various combinations, a viability assay was performed as previously described by Xu, Xiong et al. (6) with some modifications. Briefly, 10µL of ~ c. 6.6 x 107 CFU mL-1 P. aeruginosa strain PAO1 suspension were incubated (37°C, 60 mins) with 90 µL of a 2 µg/mL concentration of the various combinations and sampled every 10 mins. After incubation, the cells were diluted in deionised water and plated in Mueller hinton agar plates. Following 24 h incubation of the plates at 37°C, the percentage of viable cells was determined relative to incubation without added antibiotics. Biofilm assay: To test the susceptibility of the P. aeruginosa strain to various antibiotics in the biofilms mode of growth, overnight cultures of P. aeruginosa were diluted 1:100 into fresh medium supplemented with magnesium chloride, glucose and casamino acids. Aliquots of the dilution were dispensed into a 96 well dish and incubated (37°C, 24 h). Excess broth was removed and the number of colony forming units per milliliter (CFU/mL) of the planktonic bacteria was quantified. The biofilms were then washed and stained with 0.1% (w/v) crystal violet for 15 mins at room temperature. Following vigorous washing with water, the stained biofilms were solubilized in 30% acetic acid and the absorbance at 550nm of a 125 µL aliquot was determined in a microplate reader (Multiskan spectrum, Thermo Scientific) using 30% acetic acid in water as the blank. Aliquots of the broth prior to staining were used as an indicator of the level of planktonic growth. RESULTS AND DISCUSSION Following spray drying, the mean yield, volume weighted mean diameter and moisture content of lactoferrin powder were measured and were as follows (Table 1 and table 2); Table 1: Spray drying parameters FormulationInlet temp (°C)Outlet temp (°C)Airflow rate (L/hr)Mean yield (%)Moisture content (%) SD Lactoferrin18099 - 10060645.2 ±2.75.9 ±0.4 SD Apo Lactoferrin180100 - 10260657.8 ±1.85.7 ±0.2 Tobramycin180102 - 10460682.1 ±2.23.2 ±0.4 Lactoferrin + Tobramycin180104 - 10660687.5 ±1.43.7 ±0.2 Apo Lactoferrin + Tobramycin180103 - 10460676.3 ±2.43.3 ±0.5 Gentamicin18099 - 10260685.4 ±1.34.0 ±0.2 Lactoferrin + Gentamicin180102 - 10460687.3 ±2.13.9 ±0.3 Apo Lactoferrin + Gentamicin18099 -10360680.1±1.93.4 ±0.4 Table 2: Particle size distribution d10 d50d90 SD Lactoferrin1.384.9111.08 SD Apo Lactoferrin1.284.7911.04 SD Tobramycin1.254.9011.29 SD Lactoferrin + Tobramycin1.175.2715.23 SD Apo Lactoferrin + Tobramycin1.115.0614.31 SD Gentamicin1.406.0614.38 SD Lactoferrin + Gentamicin1.476.2314.41 SD Apo Lactoferrin + Gentamicin1.465.1511.53 The bactericidal activity of the various combinations were tested against P. aeruginosa PAO1 following a 60 minute incubation period (Figure 1 and Figure 2). While 2 µg/mL of a 1:1 combination of spray dried apo lactoferrin and Gentamicin was able to completely kill all bacterial cells within 40 mins, the same concentration was not as effective for the other antibiotic combinations. However, there was an overall reduction of bacterial cells by over 3 log units by the other combinations within 60 mins. Figure 1: Logarithmic plot of bacterial cell viability of various combinations of tobramycin and lactoferrin preparations at 2µg/mL (n = 3). Figure 2: Logarithmic plot of bacterial cell viability of various combinations of gentamicin and lactoferrin preparations at 2µg/mL (n = 3). Crystal violet staining showed that biofilm formation by P. aeruginosa PAO1 was significantly (ANOVA, p < 0.05) inhibited in the presence of the different lactoferrin preparations. Interestingly, apo lactoferrin and spray dried lactoferrin exhibited greater inhibition of both biofilm formation and biofilm persistence (Figure 2). Figure 2: Crystal violet staining of residual biofilms of P. aeruginosa following a 24hr incubation with the various combinations of antibiotics and an exposure to 48 hr formed biofilms. CONCLUSION In conclusion, combination therapy comprising of an antimicrobial peptide (lactoferrin) and an aminoglycosides (tobramycin or gentamicin) provides a feasible and alternative approach to monotherapy since the various combinations are more efficient than the respective monotherapy in the eradication of both planktonic and biofilms of P. aeruginosa. ACKNOWLEDGEMENT The authors would like to thank Mr. John Swarbrick and Friesland Campina for their generous donation of the Lactoferrin. REFERENCES 1.Hassett, D.J., Sutton, M.D., Schurr, M.J., Herr, A.B., Caldwell, C.C. and Matu, J.O. (2009), "Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways". Trends in Microbiology, 17, 130-138. 2.Trust, C.F. (2009), "Antibiotic treatment for cystic fibrosis". Report of the UK Cystic Fibrosis Trust Antibiotic Working Group. Consensus document. London: Cystic Fibrosis Trust. 3.Garcia-Contreras, L. and Hickey, A.J. (2002), "Pharmaceutical and biotechnological aerosols for cystic fibrosis therapy". Advanced Drug Delivery Reviews, 54, 1491-1504. 4.O'May, C.Y., Sanderson, K., Roddam, L.F., Kirov, S.M. and Reid, D.W. (2009), "Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions". J Med Microbiol, 58, 765-773. 5.Reid, D.W., Carroll, V., O'May, C., Champion, A. and Kirov, S.M. (2007), "Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis". European Respiratory Journal, 30, 286-292. 6.Xu, G., Xiong, W., Hu, Q., Zuo, P., Shao, B., Lan, F., Lu, X., Xu, Y. and Xiong, S. (2010), "Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa". J Appl Microbiol, 109, 1311-1318.

Powertrain optimisation in a hybrid electric bus

This paper describes the use of a formal optimisation procedure to optimise a plug-in hybrid electric bus using two different case studies to meet two different performance criteria; minimum journey cost and maximum battery life. The approach is to choose a commercially available vehicle and seek to improve its performance by varying key design parameters. Central to this approach is the ability to develop a representative backward facing model of the vehicle in MATLAB/Simulink along with appropriate optimisation objective and penalty functions. The penalty functions being the margin by which a particular design fails to meet the performance specification. The model is validated against data collected from an actual vehicle and is used to estimate the vehicle performance parameters in a model-in-the-loop process within an optimisation routine. For the purposes of this paper, the journey cost/battery life over a drive cycle is optimised whilst other performance indices are met (or exceeded). Among the available optimisation methods, Powell's method and Simulated Annealing are adopted. The results show this method as a valid alternative modelling approach to vehicle powertrain optimisation. © 2012 IEEE.

An incident at sea: The historic combat between U.S. Navy Blimp K-74 and U-Boat 134

This thesis studies the historic encounter between United States Navy airship K-74 and Nazi submarine U-134 in World War II. The Battle of the Atlantic is examined through case study of this one U-boat and its voyage. In all things except her fight with the American blimp, the patrol was perfectly typical. Looked at from start to finish, both her reports and the reports of the Allies encountered, many realities of the war can be studied. U-134 sailed to attack shipping between Florida and Cuba. She was challenged by the attack of United States Navy airship K-74 over the Florida Straits. It is the only documented instance of battle between two such combatants in history. That merits attention. Thesis finding disprove historian William Eliot Morison’s contention that the K-74 airship bombs were not dropped and did not damage the U-boat. Study of this U-boat and its antagonist broadens our understanding of the Battle of the Atlantic. It is a contribution to our knowledge of military, naval, aviation, and local history.

Radiocarbon dates of peatland initiation across the northern high latitudes

A compilation of basal dates of peatland initiation across the northern high latitudes, associated metadata including location, age, raw and calibrated radiocarbon ages, and associated references. Includes previously published datasets from sources below as well as 365 new data points.

Predicting functional disability : One year results from the Scottish Early Rheumatoid Arthritis Inception Cohort

Peer reviewed

Strategies for Coping with Multiple Biological Safety Regulations in High-Containment Laboratories

The increase in biological safety regulations and/or guidelines regarding personnel and facilities in high containment laboratories demands constant vigilance by biological safety professionals responsible for safety in these environments. Safety professionals have been faced with legislative compliance issues in the past and have developed effective management methods to cope with the demands of these requirements. Examples include the impact of the National Institutes of Health (NIH) recombinant DNA (rDNA) Guidelines and the Occupational Safety and Health Administration's (OSHA) Bloodborne Pathogens Standard. This chapter will attempt to describe seven successful strategies for management of regulatory compliance in research that are based on an overall philosophy of developing a “culture of safety”. Strategies range from interactive involvement with administration and research staff to biological safety professional development.

Contesting postliberalism: governmentality or emancipation?

This article takes the form of a debate between two theorists who work with the concept of postliberalism. Following an introduction reflecting upon what is at stake in this debate, each contribution is organised in three sections. Firstly, as an opening gambit, both authors outline their basic understanding of the concept of postliberalism. Secondly, the authors stake out their very different claims as to whether or not postliberal approaches challenge neoliberal understandings sufficiently to create new conditions for emancipa- tion or merely maintain governmentality. In the respective final sections of their con- tributions, the authors clarify the workings of postliberal approaches in policy practice.

Revisiting the Late Jurassic-Early Cretaceous of the NW South Iberian Basin: new ages and sedimentary environments

The study of the Upper Jurassic-Lower Cretaceous deposits (Higueruelas, Villar del Arzobispo and Aldea de Cortés Formations) of the South Iberian Basin (NW Valencia, Spain) reveals new stratigraphic and sedimentological data, which have significant implications on the stratigraphic framework, depositional environments and age of these units. The Higueruelas Fm was deposited in a mid-inner carbonate platform where oncolitic bars migrated by the action of storms and where oncoid production progressively decreased towards the uppermost part of the unit. The overlying Villar del Arzobispo Fm has been traditionally interpreted as an inner platform-lagoon evolving into a tidal-flat. Here it is interpreted as an inner-carbonate platform affected by storms, where oolitic shoals protected a lagoon, which had siliciclastic inputs from the continent. The Aldea de Cortés Fm has been previously interpreted as a lagoon surrounded by tidal-flats and fluvial-deltaic plains. Here it is reinterpreted as a coastal wetland where siliciclastic muddy deposits interacted with shallow fresh to marine water bodies, aeolian dunes and continental siliciclastic inputs. The contact between the Higueruelas and Villar del Arzobispo Fms, classically defined as gradual, is also interpreted here as rapid. More importantly, the contact between the Villar del Arzobispo and Aldea de Cortés Fms, previously considered as unconformable, is here interpreted as gradual. The presence of Alveosepta in the Villar del Arzobispo Fm suggests that at least part of this unit is Kimmeridgian, unlike the previously assigned Late Tithonian-Middle Berriasian age. Consequently, the underlying Higueruelas Fm, previously considered Tithonian, should not be younger than Kimmeridgian. Accordingly, sedimentation of the Aldea de Cortés Fm, previously considered Valangian-Hauterivian, probably started during the Tithonian and it may be considered part of the regressive trend of the Late Jurassic-Early Cretaceous cycle. This is consistent with the dinosaur faunas, typically Jurassic, described in the Villar del Arzobispo and Aldea de Cortés Fms.

Tipologías actuales de los trabajadores sociales en España

El objetivo del artículo es definir y explicar los tipos de profesionales del Trabajo Social existentes en España mediante una investigación descriptiva, cuantitativa y tipológica. Esta primera aproximación puede servir para iniciar la reflexión sobre la organización profesional del Trabajo Social en España y para identificar las características que definen su labor profesional. Los resultados muestran la existencia de doce tipos de profesionales del Trabajo Social en España.

La voz del otro: rompiendo con la tradición del museo

Existe una actitud predominante en el mundo de los museos según la cual las diversas culturas se presentan y se respetan de manera adecuada, dándose un diálogo efectivo entre los museos y las comunidades culturales a las que sirven. Sin embargo, el presente trabajo disiente de dicha creencia, expone sus motivos, y sugiere una trayectoria por la que los museos pueden acercarse con éxito a tales objetivos. Lo que está en juego aquí es el ethos contemporáneo e histórico de los museos y su concepto de la identidad propia, lo que interfiere con su capacidad para incluir a “los otros”, excepto en sus propios términos. El propósito del museo en este sentido es mantener su voz preeminente y su prerrogativa intelectual en sus comunicaciones con el público al que sirve. La idea de que hay múltiples voces que necesitan ser escuchadas no es un concepto fácil de contemplar para los museos, y un paso positivo en aquella dirección representaría un cambio importante con consecuencias de largo alcance.

El declive de la memoria en "Tu rostro" mañana de Javier Marías: la escritura revivida

Este trabajo tiene como objetivo estudiar los diferentes estados mentales de los personajes de las novelas de Tu rostro mañana, de Javier Marías. Estudiando las reflexiones del narrador sobre J. Deza, Peter Wheeler o Francisco Rico observamos que su decadencia mental se muestra a través de una suerte de ―presciencia‖ o lucidez momentánea que puede servir para mostrar el silencio como única tendencia de todo discurso. Desde el momento en que toda historia de ficción se cimenta sobre un discurso –no importa su cauce de presentación, ni su fuente– este es falsificado por el tiempo, la gente y cualquier otra herramienta que pueda ser utilizada para contar nada. Las conclusiones de este trabajo muestran la quimera que implica tratar de mantener una contención absoluta sobre lo acaecido, pues dicho vacío de narrativas será ocupado por una suplantación que suele ser el reverso más infame de sus actores. Es por ello que el narrador J. Deza sigue conminado a explicar sus historias, incluso allí donde uno diría que ya no puede haber ni palabras suficientes para traducir un hecho en ficción.

Receptive Vocabulary of CLIL and Non-CLIL Primary and Secondary School Learners

CLIL instruction has been reported to be beneficial for foreign language vocabulary learning since CLIL students show higher vocabulary profiles than students of their same age in traditional EFL contexts. However, to our knowledge, the receptive vocabulary knowledge of CLIL and non-CLIL learners at the end of primary and secondary education has not been examined yet. Hence, this study aims at comparing the receptive vocabulary size 79 CLIL primary learners with the receptive vocabulary knowledge of 331 non-CLIL learners at the end of primary and secondary school. Sex-based differences were also analysed. The 2k Vocabulary Levels Test (VLT) was used for the purposes of the study. Results revealed that learners’ receptive vocabulary sizes lie within the most frequent 1000 words, non-CLIL secondary school students throw better results than primary students but the differences between the secondary group and the CLIL group are not statistically significant. As for sex-based differences, we found no significant differences among the groups. These findings led us to believe that the CLIL approach offers a benefit for vocabulary acquisition since CLIL learners have been exposed to the foreign language for a shorter period of time and the results are quite similar to their non-CLIL secondary school partners.

The activity of the feminist online publications: challenges for the transmission of the feminist culture in Spain

Traditionally, big media corporations have contributed to hiding the women’s movement itself, as well as its main claims and topics of discussion (Marx, Myra y Hess, 1995; Rhode, 1995; Mendes, 2011). This has led the feminist movement to develop its own media generally print publications, usually, with a very specialized character and reduced audience. This is similar to what has occurred with quality main stream media, asthese publications have had to adapt themselves to a new communicatiion context, because of the financial crisis and  technological evolution. Feminist media has found in the Internet an excellent opportunity to access citizens and communicate their messages. , In view of this scene of change and renovation,  this article offers the results of a qualitative analysis focused on the experiences of four feminist online media sites edited in Spain: Pikaramagazine.com, Proyecto-kahlo.com, Mujeresenred.net and Laindependent.cat. Besides exploring the characteristics and content of these sites, the article pays attention to the virality of their contents spread through Facebook and Twitter. The onclusion estimates their social impact, insofar as they symbolize the specialization, diversification and dialogue promoted by the Web.