Arnot, Tom

Enright, M C

Jenkins, Toby

Pérez Esteban, Patricia

OPUS

        Citation for published version:Pérez Esteban, P, Enright, MC, Jenkins, T & Arnot, T 2014, 'Effect of nano-emulsions and bacteriophage/nano-emulsion formulations on S. aureus and P. aeruginosa growth patterns in biofilms' Paper presented at Phages2014, Oxford, UK United Kingdom, 16/09/14 - 18/09/14, .Publication date:2014Document VersionPublisher's PDF, also known as Version of recordLink to publicationPublisher RightsUnspecifiedA copy of the slides given at the conferencehttp://lpmhealthcare.com/phages-2014/event-copyright-information/The copyrights, patent rights, ownership, the right to re-use the content and the right to store, display anddistribute contents of oral and poster presentations and abstracts submitted for this event (hereafter referred toas ‘the presentations’ and ‘the abstracts’, respectively) remain with the presenter(s) and the original copyrightholder(s).University of BathGeneral rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 13. May. 20191Patricia Perez Esteban1,2, Mark C. Enright2, A.T. A. Jenkins2, Tom C. Arnot11 Department of Chemical Engineering, University of Bath2 Department of Chemistry, University of BathEffect of Nano-Emulsions and Bacteriophage/Nano-Emulsion Formulations on S. aureus and P. aeruginosa growth patterns in biofilmsPhages 2014, 18th September, Oxford, UK1. INTRODUCTION• Description of the problem of burns and burn site infections• Alternatives to antibiotics: Bacteriophages• Available technologies and implementation of the emulsification technique as an effective storage/delivery medium • Burns and burn infections1Burns are experienced by thousands of people every year in the UK with high associated costs.• Children under two years old are the most vulnerable to burn wound infections.• Minor and moderate burns caused by scalds or spillagesare the most common.• The majority of cases are domestic injuries (79%).• Complications caused by Toxic Shock Syndrome.1 “UK Burn Injury Data (1986-2007 inc.),” 2008. [Online]. Available: http://www.ibidb.org/. [Accessed: 02-Feb-2012].2 European Centre for Disease Prevention and Control, “Antimicrobial resistance surveillance in Europe 2010. AnnualReport of the European Antimicrobial Resistance Surveillance Network (EARS-Net).,” 2011.Methicillin Resistant S. aureus in Europe (2010)• Antibiotic Resistance – Evolution in Europe250% of infection cases showed antibiotic resistance3 European Centre for Disease Prevention and Control, “Antimicrobial resistance surveillance in Europe 2012. AnnualReport of the European Antimicrobial Resistance Surveillance Network (EARS-Net).,” 2013.Methicillin Resistant S. aureus in Europe (2012)50% of infection cases showed antibiotic resistance• Antibiotic Resistance – Evolution in Europe3•Increasing bacterial antibiotic resistance in Europe due to misuse or abuse•Necessity of alternatives  • Alternatives to antibiotics• Improved antibiotics – difficult to makeExample: Only Linezolid has been approved for the treatment of acuteskin infections since 2000, although Tedizolid is currently beingdeveloped.• Molecular Biology techniques to make bacteria more susceptible toantibiotics• Activated antibacterial agents• Bacteriophages4P. W. Taylor, P. D. Stapleton, and J. Paul Luzio, “New ways to treat bacterial infections.,” Drug discovery today, vol. 7, no. 21, pp. 1086-91, Nov. 2002.Alternative therapies are emerging as a consequence of the widespread antibiotic resistance4:2• Phage therapy as an alternative to antibiotics• Bacteriophages have been used against skin and wound infections, with reported success rates of up to 90% against S. aureus 7.• The advantages of bacteriophage therapy include their abundance and ecological ‘friendliness’; they can be used as a ‘phage-cocktail’, they multiply exponentially, and they do not generate unwanted side-effects 8.• There are challenges to implementing phage therapy in vivo, which may be partially addressed by modelling of population dynamics.7 Ahmad SI. 2002. Treatment of post-burns bacterial infections by bacteriophages, specifically ubiquitous Pseudomonas spp. notoriously resistant to antibiotics. Medical Hypotheses 58:327–31. 8 Hanlon GW. 2007. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. International Journal of Antimicrobial Agents 30:118–28. • Available Emulsification TechnologiesPIT EmulsificationHomogenisation2. PROJECT AIMS• Delivery of phage or ‘phage-cocktail’ to the point of infection without losing efficacy, either during delivery, or prior storage. • Use of oil-in-water nano-emulsions as a stabilising / delivery vehicle, due to their capacity to prevent virus precipitation, and to enhance transdermal penetration.• Understanding the mechanisms of interaction in a mixture containing emulsion droplets, bacteriophage, and bacteria, and the relative effects of emulsion and phage on bacterial growth.3. RESULTS• Emulsification techniques• Influence of oil-in-water Nanoemulsions on bacterial growth• Influence of oil-in-water Nanoemulsions on bacteriophage lytic activity• Product shelf-life• Bacterial Biofilms• Emulsification Techniques: PIT vs. HomogenisationFor the same formulation 80% SM Buffer (aq. phase), 15 % BrijO10 (surfactant), 5% Soybean oil (organic phase)PIT Emulsification Homogenisation after 45 min3• Emulsification Techniques: PIT vs. HomogenisationFor the same formulation 80% SM Buffer (aq. phase), 15 % BrijO10 (surfactant), 5% Soybean oil (organic phase)PIT EmulsificationHomogenisationafter 45 min• Influence of Emulsion on Bacterial Growth • Bacterial growth in more concentrated emulsion (1:1 dilution) appeared slower, showing a lag period.• RSM Analysis showed that both growth rate and carrying capacity were influenced by the concentration of emulsion droplets.Two different strainsP. P. Esteban et al. Biotechnol.Prog., 30:932–944, 2014Streptomycin in water• Influence of Emulsion on Bacterial Growth: Testing Streptomycin penetration• Bacterial growth may be influenced by attachment of emulsion droplets to theouter cell membrane, depriving bacteria of nutrients.• If so, penetration of antibiotics should also be affected.Streptomycin in the presence of emulsionP. P. Esteban et al. (Inpreparation)• Influence of Emulsion on Bacterial Growth: Different Antibiotic Mechanisms - Streptomycin• Streptomycin needs to go through and reach the ribosomes of the cell.• It binds irreversibly to the 30S ribosomal subunit.• Codon misreading and protein inhibition.• Influence of Emulsion on Bacterial Growth: Different Antibiotic Mechanisms - Vancomycin• Vancomycin sits on the cell wall surface.• It binds to the two D-ala residues.• It inhibits cell wall synthesis.• Influence of Emulsion on Bacterial Growth: Mechanism and/or Cell Wall Structure?S. aureus P. aeruginosa4• Influence of Emulsion on Bacteriophage K Lytic Activity• Bacterial concentration was dramatically reduced after the first 5 hours of treatment with bacteriophage-emulsion formulation, when compared with bacterial growth in emulsion. Re-growth is also avoided.Two different strainsP. P. Esteban et al. Biotechnol.Prog., 30:932–944, 2014• Influence of Emulsion on Bacteriophage K Lytic Activity: Possible mechanism? – Zeta PotentialBacteriophage K onlyBacteriophage K + Nano-Emulsion-15 mV-1.3 mVNano-Emulsions reduce Bacteriophage K Zeta Potential –Might eliminate electrostatic repulsion bacteria-bacteriophage• Shelf-life of Bacteriophage K-Emulsion PreparationsRelative killing effect =OD20h (control)OD20h (within phage preparation)OD20h (control)• Relative killing effect = 1 Total killing of bacteria• Relative killing effect = 0 No killing effectBacteriophage-emulsion formulations, both at room (~20oC) and cold (4oC) temperature show enhanced antibacterial activity.P. P. Esteban et al. Biotechnol.Prog., 30:932–944, 2014• Growth of Bacterial Biofilms – Crystal Violet Assay00.511.522.533.5424 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC0090700.511.522.533.5424 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC00907Growth without Emulsion Growth in EmulsionP. P. Esteban et al. (Inpreparation)• Eradication of Bacterial Biofilms – Crystal Violet AssayEradication with Phage cocktail in BufferEradication with Phage cocktail in Emulsion00.20.40.60.811.21.41.61.824 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC0090700.511.522.5324 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC00907P. P. Esteban et al. (Inpreparation)• Bacterial Biofilms – Modified Robbins Device54. MODELLING STRATEGIES• Modelling of bacterial growth – Test of existing models.• Influence of the ratio emulsion droplets : bacterial cells on growth parameters.• Proposal of a modified logistic growth model in the presence of emulsion droplets.• Infectivity models: general principles and difficulties.• Modelling bacterial growthModel Formulation ParametersLogistica=Growth rate (time-1)K=Carrying capacity (concentration)Gompertza=Growth rate (time-1)K=Carrying capacity (concentration)Richardsa=Growth rate (time-1)K=Carrying capacity (concentration)ParameterHyperbolastic H1If       0 - equivalent to the Logistic model.a=Intrinsic growth rate (time-1concentration-1)K=Carrying capacity (concentration)ParameterdS(t)dt aS(t) 1 S(t)KdS(t)dt aS(t)log KS(t)dS(t)dt aS(t) 1 S(t)KdS(t)dt 1KS(t) K  S(t)  Ka 1 t2   • Modelling bacterial growth - METHOD• In-built parameter estimation model of Matlab (lsqnonlin) not powerful enough for stiff systems of DEs. • Self-made parameter estimation algorithm using Matlab.• Multistart run for 100 random initial guesses for all growth models.• Determination of parameters, % of hits using different initial guesses, and value of total residual after fitting.• Preferred model – Simplest model.• Modelling bacterial growth - RESULTS• Logistic model yields smaller residual and all models’ parameters lead towards logistic model% of hitsValue of a• Influence of the ratio emulsion droplets : bacterial cells on growth parameters.• First moments of growth curve can be approximated to an exponential –Fitting growth rate• Use  different initial dilution factors of emulsion (vary amount of droplets per bacteria)• Proposal of a modified logistic growth model in the presence of emulsion droplets.• Growth rate is dependent on the droplet : bacteria ratio.• What about carrying capacity?dS(t)dt aS(t) 1 S(t)KdS(t)dt a0eDFS(t) 1 S(t)KNew growth model6• Infectivity models• Steps from a microscopic point of view:1. Diffusion or transport from the bulk of the solution to bacterial surface.2. Recognition and adsorption due to specific receptors on bacterial outer membrane.3. Injection of bacteriophage genetic material.4. Bacteriophage self-replication.• General mass-action law.• Alternatives to antibiotics: Bacteriophages’ mode of action• Infectivity models• General system of ODEs:dSdt Rate of appearance of bacteria by growth  Rate of disappearance of bacteria by infection dIdt Rate of appearance of bacteria  Bacterial Infection Rate  Bacterial  Inactivation Rate  Bacterial Lysis Rate dPdt Phage Inflow Rate  Phage Inactivation Rate  Phage Adsorption Rate  Phage Release of Progenie Rate • Infectivity models - ExampledS(t)dt aS(t) 1 S(t)C bS(t)P(t)dI(t)dt aI(t) 1 I(t)C bS(t)P(t) kI(t)dP(t)dt kLI(t)bS(t)P(t)mP(t)• Infectivity models - Difficulties• System of ODEs very non-linear.• Parameter estimation is not trivial.• Outcome highly dependent on initial guesses of parameters.• Having a working and reliable parameter estimation method would help elucidating the mechanisms.• Possible improvement: experimental determination of some parameters and use them as initial guesses/fixed parameters.5. CONCLUSIONS• We present a novel approach for the efficient storage and delivery of Bacteriophage K for the treatment of Staphylococcus aureusinfections. • More concentrated oil-in-water nano-emulsions had a bigger effect on bacterial growth.• The nano-emulsion-bacteriophage preparations show enhanced and stable antimicrobial activity, with reduced fluctuations of infectivity over time, when compared to a simple phage suspension.• This work demonstrates the potential for a responsive wound dressing preparation.76. ONGOING WORK• Investigation of the influence of outer cell wall properties on emulsion formulations performance in terms of growth and phage infectivity – Pseudomonas aureuginosa (Gram negative bacteria)• Experimental determination of some of the infectivity parameters in order to achieve better fitting for the modelling strategies.• Investigation of more realistic wound environments, where the presence of biofilms is determinant and critical – Use of our formulations in S. aureus and P. aureuginosa biofilms.7. FUTURE WORK• We are exploring the biological mechanisms within the system and evaluating more favourable formulations in terms of biocompatibility and cost.• We are evaluating more comprehensive approaches to modellingthe bacteriophage / emulsion / bacterial interactions.• We are moving towards a more realistic wound environment. 8. ACKNOWLEDGEMENTS• This project is funded by the Engineering and Physical SciencesResearch Council (EPSRC).• I thank my supervisors Dr Tom Arnot and Dr Toby Jenkins for theirsupport and advice.• We thank North Bristol NHS Frenchay Hospital for their support.

Effect of nano-emulsions and bacteriophage/nano-emulsion formulations on <em>S. aureus</em> and <em>P. aeruginosa</em> growth patterns in biofilms

        Citation for published version:Pérez Esteban, P, Enright, MC, Jenkins, T & Arnot, T 2014, 'Effect of nano-emulsions and bacteriophage/nano-emulsion formulations on S. aureus and P. aeruginosa growth patterns in biofilms' Paper presented at Phages2014, Oxford, UK United Kingdom, 16/09/14 - 18/09/14, .Publication date:2014Document VersionPublisher's PDF, also known as Version of recordLink to publicationPublisher RightsUnspecifiedA copy of the slides given at the conferencehttp://lpmhealthcare.com/phages-2014/event-copyright-information/The copyrights, patent rights, ownership, the right to re-use the content and the right to store, display anddistribute contents of oral and poster presentations and abstracts submitted for this event (hereafter referred toas ‘the presentations’ and ‘the abstracts’, respectively) remain with the presenter(s) and the original copyrightholder(s).University of BathGeneral rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 07. Dec. 20191Patricia Perez Esteban1,2, Mark C. Enright2, A.T. A. Jenkins2, Tom C. Arnot11 Department of Chemical Engineering, University of Bath2 Department of Chemistry, University of BathEffect of Nano-Emulsions and Bacteriophage/Nano-Emulsion Formulations on S. aureus and P. aeruginosa growth patterns in biofilmsPhages 2014, 18th September, Oxford, UK1. INTRODUCTION• Description of the problem of burns and burn site infections• Alternatives to antibiotics: Bacteriophages• Available technologies and implementation of the emulsification technique as an effective storage/delivery medium • Burns and burn infections1Burns are experienced by thousands of people every year in the UK with high associated costs.• Children under two years old are the most vulnerable to burn wound infections.• Minor and moderate burns caused by scalds or spillagesare the most common.• The majority of cases are domestic injuries (79%).• Complications caused by Toxic Shock Syndrome.1 “UK Burn Injury Data (1986-2007 inc.),” 2008. [Online]. Available: http://www.ibidb.org/. [Accessed: 02-Feb-2012].2 European Centre for Disease Prevention and Control, “Antimicrobial resistance surveillance in Europe 2010. AnnualReport of the European Antimicrobial Resistance Surveillance Network (EARS-Net).,” 2011.Methicillin Resistant S. aureus in Europe (2010)• Antibiotic Resistance – Evolution in Europe250% of infection cases showed antibiotic resistance3 European Centre for Disease Prevention and Control, “Antimicrobial resistance surveillance in Europe 2012. AnnualReport of the European Antimicrobial Resistance Surveillance Network (EARS-Net).,” 2013.Methicillin Resistant S. aureus in Europe (2012)50% of infection cases showed antibiotic resistance• Antibiotic Resistance – Evolution in Europe3•Increasing bacterial antibiotic resistance in Europe due to misuse or abuse•Necessity of alternatives  • Alternatives to antibiotics• Improved antibiotics – difficult to makeExample: Only Linezolid has been approved for the treatment of acuteskin infections since 2000, although Tedizolid is currently beingdeveloped.• Molecular Biology techniques to make bacteria more susceptible toantibiotics• Activated antibacterial agents• Bacteriophages4P. W. Taylor, P. D. Stapleton, and J. Paul Luzio, “New ways to treat bacterial infections.,” Drug discovery today, vol. 7, no. 21, pp. 1086-91, Nov. 2002.Alternative therapies are emerging as a consequence of the widespread antibiotic resistance4:2• Phage therapy as an alternative to antibiotics• Bacteriophages have been used against skin and wound infections, with reported success rates of up to 90% against S. aureus 7.• The advantages of bacteriophage therapy include their abundance and ecological ‘friendliness’; they can be used as a ‘phage-cocktail’, they multiply exponentially, and they do not generate unwanted side-effects 8.• There are challenges to implementing phage therapy in vivo, which may be partially addressed by modelling of population dynamics.7 Ahmad SI. 2002. Treatment of post-burns bacterial infections by bacteriophages, specifically ubiquitous Pseudomonas spp. notoriously resistant to antibiotics. Medical Hypotheses 58:327–31. 8 Hanlon GW. 2007. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. International Journal of Antimicrobial Agents 30:118–28. • Available Emulsification TechnologiesPIT EmulsificationHomogenisation2. PROJECT AIMS• Delivery of phage or ‘phage-cocktail’ to the point of infection without losing efficacy, either during delivery, or prior storage. • Use of oil-in-water nano-emulsions as a stabilising / delivery vehicle, due to their capacity to prevent virus precipitation, and to enhance transdermal penetration.• Understanding the mechanisms of interaction in a mixture containing emulsion droplets, bacteriophage, and bacteria, and the relative effects of emulsion and phage on bacterial growth.3. RESULTS• Emulsification techniques• Influence of oil-in-water Nanoemulsions on bacterial growth• Influence of oil-in-water Nanoemulsions on bacteriophage lytic activity• Product shelf-life• Bacterial Biofilms• Emulsification Techniques: PIT vs. HomogenisationFor the same formulation 80% SM Buffer (aq. phase), 15 % BrijO10 (surfactant), 5% Soybean oil (organic phase)PIT Emulsification Homogenisation after 45 min3• Emulsification Techniques: PIT vs. HomogenisationFor the same formulation 80% SM Buffer (aq. phase), 15 % BrijO10 (surfactant), 5% Soybean oil (organic phase)PIT EmulsificationHomogenisationafter 45 min• Influence of Emulsion on Bacterial Growth • Bacterial growth in more concentrated emulsion (1:1 dilution) appeared slower, showing a lag period.• RSM Analysis showed that both growth rate and carrying capacity were influenced by the concentration of emulsion droplets.Two different strainsP. P. Esteban et al. Biotechnol.Prog., 30:932–944, 2014Streptomycin in water• Influence of Emulsion on Bacterial Growth: Testing Streptomycin penetration• Bacterial growth may be influenced by attachment of emulsion droplets to theouter cell membrane, depriving bacteria of nutrients.• If so, penetration of antibiotics should also be affected.Streptomycin in the presence of emulsionP. P. Esteban et al. (Inpreparation)• Influence of Emulsion on Bacterial Growth: Different Antibiotic Mechanisms - Streptomycin• Streptomycin needs to go through and reach the ribosomes of the cell.• It binds irreversibly to the 30S ribosomal subunit.• Codon misreading and protein inhibition.• Influence of Emulsion on Bacterial Growth: Different Antibiotic Mechanisms - Vancomycin• Vancomycin sits on the cell wall surface.• It binds to the two D-ala residues.• It inhibits cell wall synthesis.• Influence of Emulsion on Bacterial Growth: Mechanism and/or Cell Wall Structure?S. aureus P. aeruginosa4• Influence of Emulsion on Bacteriophage K Lytic Activity• Bacterial concentration was dramatically reduced after the first 5 hours of treatment with bacteriophage-emulsion formulation, when compared with bacterial growth in emulsion. Re-growth is also avoided.Two different strainsP. P. Esteban et al. Biotechnol.Prog., 30:932–944, 2014• Influence of Emulsion on Bacteriophage K Lytic Activity: Possible mechanism? – Zeta PotentialBacteriophage K onlyBacteriophage K + Nano-Emulsion-15 mV-1.3 mVNano-Emulsions reduce Bacteriophage K Zeta Potential –Might eliminate electrostatic repulsion bacteria-bacteriophage• Shelf-life of Bacteriophage K-Emulsion PreparationsRelative killing effect =OD20h (control)OD20h (within phage preparation)OD20h (control)• Relative killing effect = 1 Total killing of bacteria• Relative killing effect = 0 No killing effectBacteriophage-emulsion formulations, both at room (~20oC) and cold (4oC) temperature show enhanced antibacterial activity.P. P. Esteban et al. Biotechnol.Prog., 30:932–944, 2014• Growth of Bacterial Biofilms – Crystal Violet Assay00.511.522.533.5424 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC0090700.511.522.533.5424 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC00907Growth without Emulsion Growth in EmulsionP. P. Esteban et al. (Inpreparation)• Eradication of Bacterial Biofilms – Crystal Violet AssayEradication with Phage cocktail in BufferEradication with Phage cocktail in Emulsion00.20.40.60.811.21.41.61.824 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC0090700.511.522.5324 h 48 h 72 hOD (570 nm)Biofilm Formation TimePAO1PAB45291BC00907P. P. Esteban et al. (Inpreparation)• Bacterial Biofilms – Modified Robbins Device54. MODELLING STRATEGIES• Modelling of bacterial growth – Test of existing models.• Influence of the ratio emulsion droplets : bacterial cells on growth parameters.• Proposal of a modified logistic growth model in the presence of emulsion droplets.• Infectivity models: general principles and difficulties.• Modelling bacterial growthModel Formulation ParametersLogistica=Growth rate (time-1)K=Carrying capacity (concentration)Gompertza=Growth rate (time-1)K=Carrying capacity (concentration)Richardsa=Growth rate (time-1)K=Carrying capacity (concentration)ParameterHyperbolastic H1If       0 - equivalent to the Logistic model.a=Intrinsic growth rate (time-1concentration-1)K=Carrying capacity (concentration)ParameterdS(t)dt aS(t) 1 S(t)KdS(t)dt aS(t)log KS(t)dS(t)dt aS(t) 1 S(t)KdS(t)dt 1KS(t) K  S(t)  Ka 1 t2   • Modelling bacterial growth - METHOD• In-built parameter estimation model of Matlab (lsqnonlin) not powerful enough for stiff systems of DEs. • Self-made parameter estimation algorithm using Matlab.• Multistart run for 100 random initial guesses for all growth models.• Determination of parameters, % of hits using different initial guesses, and value of total residual after fitting.• Preferred model – Simplest model.• Modelling bacterial growth - RESULTS• Logistic model yields smaller residual and all models’ parameters lead towards logistic model% of hitsValue of a• Influence of the ratio emulsion droplets : bacterial cells on growth parameters.• First moments of growth curve can be approximated to an exponential –Fitting growth rate• Use  different initial dilution factors of emulsion (vary amount of droplets per bacteria)• Proposal of a modified logistic growth model in the presence of emulsion droplets.• Growth rate is dependent on the droplet : bacteria ratio.• What about carrying capacity?dS(t)dt aS(t) 1 S(t)KdS(t)dt a0eDFS(t) 1 S(t)KNew growth model6• Infectivity models• Steps from a microscopic point of view:1. Diffusion or transport from the bulk of the solution to bacterial surface.2. Recognition and adsorption due to specific receptors on bacterial outer membrane.3. Injection of bacteriophage genetic material.4. Bacteriophage self-replication.• General mass-action law.• Alternatives to antibiotics: Bacteriophages’ mode of action• Infectivity models• General system of ODEs:dSdt Rate of appearance of bacteria by growth  Rate of disappearance of bacteria by infection dIdt Rate of appearance of bacteria  Bacterial Infection Rate  Bacterial  Inactivation Rate  Bacterial Lysis Rate dPdt Phage Inflow Rate  Phage Inactivation Rate  Phage Adsorption Rate  Phage Release of Progenie Rate • Infectivity models - ExampledS(t)dt aS(t) 1 S(t)C bS(t)P(t)dI(t)dt aI(t) 1 I(t)C bS(t)P(t) kI(t)dP(t)dt kLI(t)bS(t)P(t)mP(t)• Infectivity models - Difficulties• System of ODEs very non-linear.• Parameter estimation is not trivial.• Outcome highly dependent on initial guesses of parameters.• Having a working and reliable parameter estimation method would help elucidating the mechanisms.• Possible improvement: experimental determination of some parameters and use them as initial guesses/fixed parameters.5. CONCLUSIONS• We present a novel approach for the efficient storage and delivery of Bacteriophage K for the treatment of Staphylococcus aureusinfections. • More concentrated oil-in-water nano-emulsions had a bigger effect on bacterial growth.• The nano-emulsion-bacteriophage preparations show enhanced and stable antimicrobial activity, with reduced fluctuations of infectivity over time, when compared to a simple phage suspension.• This work demonstrates the potential for a responsive wound dressing preparation.76. ONGOING WORK• Investigation of the influence of outer cell wall properties on emulsion formulations performance in terms of growth and phage infectivity – Pseudomonas aureuginosa (Gram negative bacteria)• Experimental determination of some of the infectivity parameters in order to achieve better fitting for the modelling strategies.• Investigation of more realistic wound environments, where the presence of biofilms is determinant and critical – Use of our formulations in S. aureus and P. aureuginosa biofilms.7. FUTURE WORK• We are exploring the biological mechanisms within the system and evaluating more favourable formulations in terms of biocompatibility and cost.• We are evaluating more comprehensive approaches to modellingthe bacteriophage / emulsion / bacterial interactions.• We are moving towards a more realistic wound environment. 8. ACKNOWLEDGEMENTS• This project is funded by the Engineering and Physical SciencesResearch Council (EPSRC).• I thank my supervisors Dr Tom Arnot and Dr Toby Jenkins for theirsupport and advice.• We thank North Bristol NHS Frenchay Hospital for their support.

https://purehost.bath.ac.uk/ws/files/130363182/Phages_2014_Patricia_P.pdf

Effect of nano-emulsions and bacteriophage/nano-emulsion formulations on <em>S. aureus</em> and <em>P. aeruginosa</em> growth patterns in biofilms

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