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.

Eastern Christianity and politics in the Twenty-First Century

This book provides an up-to-date, comprehensive overview of Eastern Christian churches in Europe, the Middle East, America, Africa, Asia and Australia. Written by leading international scholars in the field, it examines both Orthodox and Oriental churches from the end of the Cold War up to the present day. The book offers a unique insight into the myriad church-state relations in Eastern Christianity and tackles contemporary concerns, opportunities and challenges, such as religious revival after the fall of communism; churches and democracy; relations between Orthodox, Catholic and Greek Catholic churches; religious education and monastic life; the size and structure of congregations; and the impact of migration, secularisation and globalisation on Eastern Christianity in the twenty-first century.

The Romanian Orthodox Church

At first glance, the nationalist ideology of the French Revolution seems to have had little impact on the Orthodox Church in Romanian-speaking territories. Romanians were the predominant inhabitants of the principalities of Wallachia and Moldavia and the neighboring territories of Transylvania (including Crişana, Maramureş and Banat), Bukovina, Bessarabia, and Dobrudja. The majority of ethnic Romanians belonged to the Orthodox faith while their communities were at the intersection of geopo liti cal interests of the Rus sian, Ottoman, and Habsburg empires. In 1859 the Principalities of Wallachia and Moldavia (known as the Old Kingdom between 1866 and 1918) united into a single state under the rule of a local prince. The term "Romania" began to be used by the new state in its of cial documents in 1862. Two years later, the state supported the declaration of a Romanian autocephalous (in de pen dent) church that was recognized by the Ecumenical Patriarchate in 1885. As an integrative part of the Orthodox commonwealth, the church was situated between the competing jurisdictions of the Ecumenical Patriarchate and the Rus sian Orthodox Church, while its declaration of autocephaly followed a pattern in the spread of national churches in Southeastern Europe. From the Treaty of Kuchuk Kainardji of 1774 to the beginning of the Greek War for In de pen dence in 1821, the Romanian principalities were under the suzerainty of the Ottoman Empire, which had full control of their po liti cal and economic affairs. The sultan appointed princes, and the Porte determined their po liti cal and judicial status. The princes were drawn from the "Phanariots," and were directly appointed by the Porte from preponderantly Greek elite rather than the Romanian local elite, the boyars (boieri).1 In each principality, the church was headed by a metropolitan who was under the direct jurisdiction of the Ecumenical Patriarchate. That religion mattered to local population as a means of social cohesion was suggestively depicted by Anatole de Demidoff, an En glish traveler in the region in 1837. Arriving in Bucharest, the capital of Wallachia, he claimed that: I know of no city in Europe in which it is possible to find more agreeable society, or in which there is a better tone, united with the most charming gaiety⋯. Religion, which is here of the schismatic Greek creed, does not, properly speaking, hold any great empire over the minds of the Wallachian people, but they observe its outward forms, and particularly the austerities of fasting, with scrupulous exactitude. The people are seen to attend divine ser vice with every sign of respect, and the great number of churches existing in Wallachia, bear witness to the ardent zeal with which outward worship is honored.2 The Romanian Orthodox Church was a national institution, closely linked to social, economic, and po liti cal structures. In most cases, Orthodox hierarchs were appointed from the families of boyars, thus ensuring a close relationship with the state authorities and its policies. As one of the largest landowners in the principalities, the church had a prime role in administrating healthcare and education. Although the majority of the clergy was uneducated, it dispensed both ecclesiastical and civil justice and in many cases worked closely with boyars in local administration.3 The lower clergy not only contributed directly to the economy but also benefited from tax privileges. Some small villages had an unusually high proportion of clergy in comparison to the overall population. For example, in 1810, Stənisləveşti, a village in the south of Wallachia, was composed of eleven houses and had two priests, five deacons, and three cantors; similarly, the Frəsinet village of nineteen houses had two priests and five deacons.4 Although these cases were exceptional, they indicate both the economic value of being a member of the clergy and the wider canonical dimension of church jurisdiction. The special status of the clergy was reflected not only at lower but also at higher levels. Bishops and metropolitans engaged with state policy and in many cases opposition to the authorities led to the loss of a spiritual seat. The metropolitan of each principality worked with the prince and was president of the divan, the gathering of all boyars. He held the right to be the first person to comment on state policy and to make recommendations when the prince was absent. The metropolitan replaced the prince when the principality had no political ruler, such as in the cases of Metropolitan Veniamin Costachi of Moldavia in 1806 and Metropolitan Dositei Filitti of Wallachia, while the bishops of Buzəu and Argeş were members of the provisional government during the Rus sian occupation of the principalities in 1808. The higher clergy had both religious and political prerogatives in relation to foreign powers as evident in their heading of the boyars' delegation to peace negotiation between the Rus sian and Ottoman empires at Focşani in 1772 and addressing memoranda to the Austrian and Rus sian governments in 1802.5 The primary role of the church in the principalities of Moldavia and Wallachia was paralleled by the national mobilization of Orthodox communities in the neighboring territories that had Romanian inhabitants. Although throughout the region Orthodox communities were incorporated into church structures as part of the Habsburg, Austrian or Rus sian empires, the nineteenth century was characterized by the leadership's search for political autonomy and the building of a Romanian national identity. The Orthodox communities outside the Old Kingdom maintained relations with the faithful in principalities across the Carpathian Mountains and the Dniester River and sought support in their struggle for political and religious rights.