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.

Broadly tunable CW green-to-red laser source based on frequency doubling of a quantum-dot external cavity diode laser in a PPKTP waveguide

Here we present a compact tunable all-room-temperature frequency-doubling scheme, using a periodically poled potassium titanyl phosphate (PPKTP) waveguide and a QD-ECDL. A broad wavelength tunability of the second harmonic generated light (SHG) in the spectral region between 567.7 and 629.1 nm was achieved, with maximum conversion efficiencies in range of 0.34%-7.9%. The maximum output power for the SHG light was 4.11 mW at 591.5 nm, achieved for 52 mW of launched pump power at 1183 nm, resulting in a conversion efficiency of 7.9%.

Orange light generation from a PPKTP waveguide end pumped by a cw quantum-dot tunable laser diode

A compact all-room-temperature frequency-doubling scheme generating cw orange light with a periodically poled potassium titanyl phosphate waveguide and a quantum-dot external cavity diode laser is demonstrated. A frequency-doubled power of up to 4.3 mW at the wavelength of 612.9 nm with a conversion efficiency exceeding 10% is reported. Second harmonic wavelength tuning between 612.9 nm and 616.3 nm by changing the temperature of the crystal is also demonstrated. © Springer-Verlag 2010.

Green-to-red tunable SHG of a quantum-dot laser in a PPKTP waveguide

Quasi-phase-matching is an important and widelyused technique in nonlinear optics enabling efficient frequency up-conversion. However, since its introduction almost half a century ago, this technique is well developed for near infrared (IR) but is intrinsically limited in spectral tunability in the visible range by the strict conditions set by the spatial modulation which compensates the momentum mismatch imposed by the dispersion. Here, we provide a fundamental generalization of quasi-phase-matching based on the utilization of a significant difference in the effective refractive indices of the high- and low-order modes in multimode waveguides. This concept enables to match the period of poling in a very broad wavelength range and opens up a new avenue for an order-ofmagnitude increase in wavelength range for frequency conversion from a single crystal. Using this approach, we demonstrate an all-room-temperature continuous-wave (CW) second harmonic generation (SHG) with over 60 nm tunability from green to red in a periodically-poled potassium titanyl phosphate (PPKTP) waveguide pumped by a single broadly-tunable quantumdot laser diode. © 2012 by Astro, Ltd.

Orange-to-red tunable picosecond pulses by frequency doubling in a diode-pumped PPKTP waveguide

A compact picosecond all-room-temperature orange-to-red tunable laser source in the spectral region between 600 and 627 nm is demonstrated. The tunable radiation is obtained by second-harmonic generation in a periodically poled potassium titanyl phosphate (PPKTP) multimode waveguide using a tunable quantum-dot external-cavity mode-locked laser. The maximum second-harmonic output peak power of 3.91 mW at 613 nm is achieved for 85.94 mW of launched pump peak power at 1226 nm, resulting in conversion efficiency of 4.55%. © 2013 Optical Society of America.

73 nm wavelength tuning from a frequency-doubled quantum-dot laser in PPKTP waveguides

A compact all-room-temperature CW 73-nm tunable laser source in the visible spectral region (574nm-647nm) has been demonstrated by frequency-doubling of a broadly-tunable InAs/GaAs quantum dot external-cavity diode laser in periodically-poled potassium titanyl phosphate waveguides with a maximum output power in excess of 12mW and a maximum conversion efficiency exceeding 10%. Three waveguides with different cross-sectional areas (4×4μm2, 3×5μm2 and 2x6μm2) were investigated. Introduction - Development of compact broadly tunable laser sources in the visible spectral region is currently very attractive area of research with applications ranging from photomedicine and biophotonics to confocal fluorescence microscopy and laser projection displays. In this respect, semiconductor lasers with their small size, high efficiency, reliability and low cost are very promising for realization of such sources by frequency­doubling of the infrared light in nonlinear crystal waveguides. Furthermore, the wide tunability offered by quantum-dot (QD) external-cavity diode lasers (ECDL), due to the temperature insensibility and broad gain bandwidth [1,2], is very promising for the development of tunable visible laser sources [3,4]. In this work we show a compact green-to-red tunable all­room-temperature CW laser source using a frequency-doubled InAs/GaAs QD-ECDL in periodically-poled potassium titanyl phosphate (PPKTP) crystal waveguides. This laser source generates frequency-doubled light over the 574nm-647nm wavelength range utilizing the significant difference in the effective refractive indices of high-order and low-order modes in multimode waveguides [3]. Experimental results - Experimental setup used in this work was similar to that described in [3] and consisted of a QD gain chip in the quasi­Littrow configuration and a PPKTP waveguide. Coarse wavelength tuning of the QD-ECDL between 1140 nm and 1300 nm at 20°C was possible for pump current of 1.5 A. The laser output was coupled into the PPKTP waveguide using an AR-coated 40x aspheric lens (NA ~ 0.55). The PPKTP frequency-doubling crystal (not AR coated) used in our work was 18 mm in length and was periodically poled for SHG (with the poling period of ~ 11.574 11m). The crystal contained 3 different waveguides with cross-sectional areas of ~ 4x4 11m2, 3x5 11m2 and 2x6 11m2. Both the pump laser and the PPKTP crystal were operating at room temperature. The waveguides with cross-sectional areas of 4x411m2, 3x511m2 and 2x611m2 demonstrated the tunability in the wavelength ranges of 577nm - 647nm, 576nm -643nm and 574nm - 641nm, respectively, with a maximum output power of 12.04mW at 606 nm Conclusion - We demonstrated a compact all-room-temperature broadly­tunable laser source operating in the visible spectral region between 574nm and 647nm. This laser source is based on second harmonic generation in PPKTP waveguides with different cross-sectional areas using an InAs/GaAs QD-ECDL References [I] E.U. Rafailov, M.A. Cataluna, and W. Sibbett, Nat. Phot. 1,395 (2007). [2] K.A. Fedorova, M.A. Cataluna, I. Krestnikov, D. Livshits, and E.U. Rafailov, Opt. Express 18(18), 19438-19443 (2010). [3] K.A. Fedorova, G.S. Sokolovskii, P.R. Battle, D.A. Livshits, and E.U. Rafailov, Laser Phys. Lett. 9, 790-795 (2012). [4] K.A. Fedorova,G.S. Sokolovskii, D.T. Nikitichev, P.R. Battle, I.L. Krestnikov, D.A. Livshits, and E.U. Rafailov, Opt. Lett. 38(15), 2835-2837 (2013) © 2014 IEEE.

574-647 nm wavelength tuning by second-harmonic generation from diode-pumped PPKTP waveguides

We present a compact, all-room-temperature continuous-wave laser source in the visible spectral region between 574 and 647 nm by frequency doubling of a broadly tunable InAs/GaAs quantum-dot external-cavity diode laser in a periodically poled potassium titanyl phosphate crystal containing three waveguides with different cross-sectional areas (4 × 4, 3 × 5, and 2 μm × 6 μm). The influence of a waveguide's design on tunability, output power, and mode distribution of second-harmonic generated light, as well as possibilities to increase the conversion efficiency via an optimization of a waveguide's cross-sectional area, was systematically investigated. A maximum output power of 12.04 mW with a conversion efficiency of 10.29% at 605.6 nm was demonstrated in the wider waveguide with the cross-sectional area of 4 μm × 4 μm.

Tunable single- and dual-wavelength SHG from diode-pumped PPKTP waveguides

A compact, all-room-temperature, widely tunable, continuous wave laser source in the green spectral region (502.1–544.2 nm) with a maximum output power of 14.7 mW is demonstrated. This was made possible by utilizing second-harmonic generation (SHG) in a periodically poled potassium titanyl phosphate (PPKTP) crystal waveguide pumped by a quantum-well external-cavity fiber-coupled diode laser and exploiting the multimode-matching approach in nonlinear crystal waveguides. The dual-wavelength SHG in the wavelength region between 505.4 and 537.7 nm (with a wavelength difference ranging from 1.8 to 32.3 nm) and sum-frequency generation in a PPKTP waveguide is also demonstrated.