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.

Policy through procurement:introduction of digital signal process (DSP) hearing aids into the English NHS

Despite being a major user of many technologies and innovations, the healthcare sector's role and influence as a procurer of technologies has been poorly represented by the literature and consequently is not fully understood. Providing a practical example of the introduction of digital signal process (DSP) hearing aids in to the English NHS, this paper discusses the role of public sector procurement agencies in the uptake of technologies from the private sector and their adoption by the public sector. Employing a system of innovation (SI) approach, the paper highlights the need for policy-makers to adopt a dynamic as well as systemic perspective that recognises the shifting roles, responsibilities and interactions of key stakeholders throughout the innovation process.

Supply management:is it a discipline?

Purpose – To examine management literature for guidance on what constitutes a discipline. To examine supply management publications to determine whether the field constitutes a discipline or an emerging discipline. To contribute a structured evaluation to the body of supply management theory/discipline development knowledge. Design/methodology/approach – Literature review of what constitutes a discipline and an initial assessment of whether supply management is a discipline. Development of research questions used to design tests, using combinations of qualitative pattern matching, journal quality rankings, and social science citations index impact factor. Application of the tests, to evaluate field coherence, quality and the existence of a discipline-debate, to determine whether supply management is an emerging discipline. Findings – An initial literature review finds supply management not to be a discipline, as the field lacks quality of theoretical development and discussion, and coherence. Tests for increasing evidence of coherence, quality and impact yield positive results, indicating that supply management is progressing in its theoretical development. The test findings combined with the existence of the start of a discipline-debate indicate that supply management should be judged to be an emerging discipline. Originality/value – Drawing from the management literature, the paper provides a unique structured evaluation of the field of supply management, finding it not to be a discipline, but showing evidence of being an emerging discipline.

Promoting competitive markets:the role of public procurement

The study presented here examines how public procurement agencies address establishing and maintaining competitive markets; a topic still in its academic infancy. Cases are used to address impediments and improve understanding of strategic priorities in managing for competitive markets. Public policy academics have observed many competing policies in the wider public sector. Specifically, this paper identifies a need for research on supplier incentives at a market level, on the post contract management of suppliers and as an important sub-set, key supplier relationship management, along with professional development.

Reflecting on time, timing and timeliness in public sector supply networks

Despite recent research on time (e.g. Hedaa & Törnroos, 2001), consideration of the time dimension in data collection, analysis and interpretation in research in supply networks is, to date, still limited. Drawing on a body of literature from organization studies, and empirical findings from a six-year action research programme and a related study of network learning, we reflect on time, timing and timeliness in interorganizational networks. The empirical setting is supply networks in the English health sector wherein we identify and elaborate various issues of time, within the case and in terms of research process. Our analysis is wide-ranging and multi-level, from the global (e.g. identifying the notion of life cycles) to the particular (e.g. different cycle times in supply, such as daily for deliveries and yearly for contracts). We discuss the ‘speeding up’ of inter-organizational ‘e’ time and tensions with other time demands. In closing the paper, we relate our conclusions to the future conduct of the research programme and supply research more generally, and to the practice of managing supply (in) networks.

Factors positively influencing academically successful, highly motivated African -American high school students

The purpose of this study is two-fold: (1) to investigate the common factors positively influencing academically successful, highly motivated African-American high school students in their educational pursuits; and (2) to determine ways in which educators can facilitate an increased positive impact on minority students' achievement motivation and transition to higher education. ^ Three sources of data inform the qualitative study. Short autobiographical essays address the topic of antecedent factors influencing the students' selection of an academic program of study. Individual interviews using a semi-structured format elicit discussion of prior experiences and influences related to the research sub-questions. Focus group interviews elicit discussion of emergent patterns perceived by the researcher from the autobiographical essays and individual interviews. ^ Results indicate a generic model for success for high achieving African-American students. Students prioritize education as a means to success in life. They receive strong support for educational aspirations from at least one adult within the home. The students are focused on a professional career in the future. The students realize and desire the financial benefits of academic success. The students desire academic challenge. Students have feelings of control over their academic choices; however, they frequently seek the counsel of family members. Prior experience in honors, gifted, or magnet programs did not meet needs. ^ Recommendations for improved educational experiences include: Increase teacher and counselor responsiveness to students. Provide classroom activities which afford more interaction among the students and teacher and relate curriculum to topics of interest. Increase involvement of parents and Black community members in establishing goals and visions of opportunity. Recognize and revere diversity among students within the classroom environment. Set a climate that assumes more individual student responsibility and sets higher academic expectations. ^ The factors influencing academically successful, highly motivated African-American high school students echo the patterns of experiences of many groups. Pride in achievement, emotional support for efforts, expectations of success, independence in thought, and consternation with mediocrity and malaise drive the students to excel academically just like all others who succeed. ^

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

Diferenciais de mortalidade adulta por nível de escolaridade no Brasil e regiões

The study of mortality by various differentials has been an important tool to guide public health policies, due to better describing the events of deaths in a population. This research aims to seek disparities in mortality according to educational level, sex and adulthood in large Brazilian regions and consequently for Brazil as a whole. A vast literature has shown that people with more education tend to have lower risk of death. Studies on inequalities in mortality by level of education in Brazil are still very specific and has still known very little about Brazil about mortality according to educational level, due to lack of information about the well-filled school in the records of deaths arising from the Mortality Information System (MIS) of the Ministry of Health. This data source has shown improvement in the coverage of sub reports in the last decade, however, it has still perceived negligence in completing the question regarding schooling of death (about 30% of registered deaths in 2010 to Brazil, Given this scenario, this work contributes to the national literature on the behavior of adult mortality differentials having as proposed, using data from the new variable mortality of the 2010 Census (CD 2010), assuming the characteristics of education of the head the household for deaths occurring in the same. It is therefore considered that the probability of mortality is homogeneous within the household. Events of deaths were corrected only for the records come from households where the head possessed levels of schooling and Instruction Elementary Education No Incomplete and Primary Education and Secondary Education Complete Incomplete through the Generations Extinct Adjusted method. With deaths already corrected, probabilities of death were calculated between 15 and 60 years life, as well as tables by sex and level of education to all regions of Brazil. No que se refere às probabilidades de morte por idade, nas idades entre 15 e 60 anos as maiores probabilidades seguem um gradiente, maior probabilidade para os menos escolarizados. Results corroborate the literature, the more educated the population is, the greater the life expectancy. In all Brazilian regions, life expectancy of the female population is greater than that of men at all levels of schooling. With respect to probabilities of death by age between the ages of 15 and 60 years the most likely follow a gradient, most likely to the least educated. At older ages (from 70 years), this behavior has presented another pattern, the lowest level of education has the lowest odds in the regions, North, Northeast, South and Midwest, except in the Southeast region

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.

What is this thing called Historical Archaeology?

Historical archaeology, in its narrow temporal sense -as an archaeology of the emergence and subsequent evolution of the Modern world- is steadily taking pace in Spanish academia. This paper aims at provoking a more robust debate through understanding how Spanish historical archaeology is placed in the international scene and some of its more relevant particularities. In so doing, the paper also stresses the strong links that have united historical and prehistorical archaeology since its inception, both in relation to the ontological, epistemological and methodological definition of the first as to the influence of socio-political issues in the latter. Such reflection is partly a situated reflection from prehistory as one of the paper’s authors has been a prehistorian for most of her professional life.