GB2406053A

GB2406053A - Antimicrobial compounds

Info

Publication number: GB2406053A
Application number: GB0321130A
Authority: GB
Inventors: Gordon Nelson; Valerie Edwards-Jones; Rachael Buck; Amanda Jane Lyne
Original assignee: Micap PLC
Current assignee: Micap PLC
Priority date: 2003-09-10
Filing date: 2003-09-10
Publication date: 2005-03-23
Anticipated expiration: 2023-09-10
Also published as: GB2406053B; GB0321130D0

Abstract

A composition for use as an antimicrobial medicament comprising at least one essential oil and a fungal cell or fungal cell fragment, wherein the essential oil is encapsulated by the fungal cell or cell fragment. The composition is preferably used for the treatment of Staphylococcus infection, particularly methicillin resistant s. aureus (MRSA). Wound dressings comprising the composition are also disclosed. An alternative embodiment provides a composition comprising a biocidal compound and at least one essential oil, and a further embodiment provides a composition comprising a first and a second essential oil.

Description

ANTIMICROBIAL COMPOSITION

The present Invention relates to antimicrobial compositions and methods of using the same In particular the present nvenbon relates to anDmcrobal compositions and methods fomnhibiting microbial development in wounds Control of infection in wounds Is mainly achieved by fungicides, bactericdes and/or antibiotics.

However, these synthetic chemicals can be toxic in high concentrations.

Furthermore, the development of resistance to these biocides has been observed In some strains of microorganisms Consequently, many fungicides and bactericdes are being phased out by regulating agencies Despite major advances in wound management, infection sell remains an important factor in wound healing For instance, In burns approximately 75% of deaths are due to complications with sepsis from wound infection (1) Among other adverse effects, infection delays healing, contributes to graft failure and can Increase the depth of a burn. Approximately 30% of burn wounds become colonized with Staphylococcus aureus (2) and outbreaks of methicilln-resistant S. aureus (MRSA) have created mayor problems for burn units and intensive care units In terms of cross Infection and rehabilitation of the pabent due to imposed barrier nursing (3) Some MRSA strains, such as epidemic MRSA (EMRSA) have the ability to spread rapidly among patients and the dominant clonal EMRSA types 15 and 16 are problematic In the UK (4,5).

Staphylococci are an example of common bacteria that live on the skin and mucous membranes (e.g In the nose) of humans About 15-40 per cent of healthy humans are carriers of S. aureus, that is, they have the bacteria on their skin without any active Infection or disease (colonsaton) S aureus is the most pathogenic species of the Genus as they can cause potentially fatal diseases and currently major concern focuses around their increasing resistance to anbbotcs In the USA and the UK 90% of S. aureus isolates are resistant to penicillin G and Incidence of methiclln resistance (MRSA) Is rising exponentially Vancomycn Is one of the few effective systemic anDbotics available for treatment, however increased inhibitory concentrations (intermediate resistance) has been reported (Vancomycin intermediate Staphylococcus aureus VlSA)and there is mayor concern that total antibiotic resistant strains may emerge in the Immediate future (6). However, because of its toxicity and the threat of resistance its use is controlled.

To date topical anti-microbial therapy Is the single most important component of wound care to prevent infection (7) and In hosptalised burns patients, Flamazine _ Is by far the most frequently used topical prophylactic agent (8) but does not always penetrate into the wound (9) and cannot be used to eradicate carriage from the patient or the environment. Thus means of preventing infection, reducing colonization of the patient by microorganisms and thus reducing the need for administered antibiotics is needed.

It is an object of the present invention to alleviate or overcome one or more of the problems associated with the prior art and/or to provide an improved antimicrobial composition. It Is a further object of the invention to provide an improved method for inhibiting or preventing microbial development In wounds or other lesions.

In accordance with a first aspect of the present Invention there is provided a composition for use as a medcament, which composition comprises at least one essential oil and a fungal cell or fungal cell fragment wherein molecules of the at least one essential oil are encapsulated or partially encapsulated by the fungal cell or fungal cell fragment.

In accordance with a further aspect of the present nvenbon, there Is provided a composition comprising at least one essential oil and at least one bocdaland/or anDbotic compound.

The bocdal compound may be a fungicide and/or a bactericide The biocidal compound may be selected from phenols and cresols, acids and esters, alkalis, chlorine release agents, iodine compounds, quaternary ammonium compounds, bguandes, damidnes, aldehydes, alcohols, heavy metal derivatives, vapour phase disinfectants, sulphates and nitrites, for example Preferably, the biocidal compound is a bactericide More preferably, the bactericide Is tnclosan (obtainable from Cambiochem California, USA of EMD Biosciences Inc. an affiliate of Merck, Germany). Other preferred brocades include chlorhexdne, povidone iodine and/or silver sulphadiazine, for example.

Preferred antibiotics include mupirocin, fustian and gentamicin, for example.

In accordance with a further aspect of the present invention, there Is provided a composition comprising an essential oil, a biocdal and/or antibiotic compound and a fungal cell or fungal cell fragment, wherein molecules of at least one of the essential oil or biocidal/antibiotc compound are encapsulated or partially encapsulated by the fungal cell or fungal cell fragment.

In accordance with a further aspect of the present invention, there Is provided a composition comprising two or more essential oils and a fungal cell or fungal cell fragment wherein molecules of at least one essential oil is encapsulated or partially encapsulated by the fungal cell or fungal cell fragment.

In accordance with a further aspect of the present Invention, there Is provided a therapeutic formulation comprising a composition as described hereinabove. The formulation may comprise one or more excpents In accordance with a further aspect of the present Invention there Is provided the use of a composition for the manufacture of a medcament for the treatment or prophylaxis of microbial infection, the composition comprising at least one essential oil and a fungal cell or fungal cell fragment, wherein molecules of the essential oil are encapsulated or partially encapsulated by the fungal cell or fungal cell fragment.

Preferably, the composition Is for the treatment or prophylaxis of Staphylococcus infection. Strains of staphylococcus Include S aureus, S ep/derm/d/s, S. saprophybcus, S. haemolybcus, Methicllin sensitive S. aureus (MSSA), Methclln resistant S. aureus (MRSA) and Epidemic methcilln resistant S. aureus (EMRSA) More preferably, the composition is for the treatment of MRSA.

In accordance with a further aspect of the present nvenbon there is provided a method of treating or preventing a microbial nfechon In a subject composing admnstenng to a subject a composition as described hereinabove.

In accordance with a further aspect of the present Invention there is provided a composition comprising: a) a first essential oil comprising Manuka and a second essential oil comprising one or more essential oil selected from the group comprising Geranium, Lavender, Lemongrass, and Tea tree, or b) a first essential oil comprising Geranium and a second essential oil comprising one or more essential oil selected from the group composing Manuka, Lavender, Lemongrass, and Tea tree; or c) a first essential oil comprising Lemongrass and a second essential oil comprising one or more essential oil selected from the group comprising Geranium, Lavender, Manuka, and Tea tree; or d) a first essential oil comprising Lavender and a second essential oil comprising one or more essential oil selected from the group compnsng Geranium, Manuka, Lemongrass, and Tea tree, or e) a first essential oil comprising Tea tree and a second essential oil comprising one or more essential oil selected from the group comprising Geranium, Lavender, Lemongrass, and Manuka.

Essenbal oils are complex mixtures of odorous, steam volatile or extractable organic compounds, which are synthessed by many types of plant Essential oils can be found in venous parts of a plant, such as the leaves, stem, flowers, cell organelles, fruit, roots, seeds and bark etc Generally, the pnncpal constituents are aromatic compounds. Each oil may comprise 100-300 compounds Essential oils most abundant components Include one or more Mono-, di- and sesqui- terpenods (mevalonc acid derived constituents); phenylpropanoids; alkanes (and alkane derivatives, such as alcohols, aldehydes, and carboxylc acids), alkenes, alkynes and denvatves thereof Essential oils are typically mixtures of organic aromatic and other compounds that are extractable from plant material by methods such as steam distillation, cold pressing, CO2 extraction or extraction with organic solvents or any other means known to the person skilled in the art.

Essential oils for use In the present invention include but are not limited to extracts from Bay (Pimenta recemosa), Bergamot (Citrus bergamia); Cardamom (Eleffaria cardamom); Cedarwood (Cedrus deodara and Juniperus Virginians); Cinnamon leaf (C/nnamomum zellamcum Ceylon); Clove or clove bud (Eugenia caryophyllata Madagascar extra; Syzygium aromas/cum L./Eugema aromas/cum L);Cumin seed (Cummum cymmum); Eucalyptus (Eucalyptus globules & radiate); Geranium (Pelargon/um graveolens Madagascar bourbon); Grapefruit (Citrus paradises; Lavender (Lavendula officinal/s France);Lemongrass (Cymbopogon citrates), Manuka (Leptospermum scoparium); Marjoram (Onganum majorana); Origanum (Origanum vulgarel Cymbopogon martin/); Palmarosa (Origanum heracleoticum), Patchouli (Pogostemon cablin E India dark); Peppermnt (Mentha piper/la); Rosemary (Rosmarinus officinal/s); Rosewood (Aniba rosaeodora); Sage (Salvia tribola); Sandalwood (Amba rosaeodora); Savory (Satureia thymbra), Tea Tree (Melaleuca altemifolalLeptospermum peterson//; Thyme (Thymus capitus). Other essential oils useful in the present invention include Sandal oil, KapurTuls'oil, and Ropan oil Preferably, compositions according to the present invention comprise one or more essential oils from the group comprising Manuka, Geranium, Lavender, Lemongrass and Tea tree. More preferably, the compositions of the present nvenbon compose two or more essential oils selected from the group comprising Manuka, Geranium, Lavender, Lemongrass and Tea tree More preferably still, the composition of the present inventor comprises one or more of the following combinations of essential oils Manuka + Geranium, Geranium + Lavender, Manuka + Lavender, Geranium + Lemongrass, Manuka + Lemongrass, Geranium + Tea tree, Manuka + Tea tree, Lavender + Lemongrass, Lemongrass + Tea tree and Lavender + Tea tree.

Other common chemical constituents of essential oils are citral (geranial and neral isomers), limonene, linalyl acetate and estragole (methyl chavcol), mono-, sesqu'- and d'- terpenods (mevalonic acid- derived constituents); phenylpropanods (cinnamic acid-derved compounds) and alkane derivatives (alkanes, alkenes, alkynes, alkanols, alkanals, alkanoic acids: mostly acetogenns).

It Is understood that the term "essential oil" as used herein includes the naturally occumng extractable plant oils, mixtures thereof, or one or more of the components found in extractable plant oils, whether naturally or artificially synthesized. The term also includes derivatives and analogues of the components found in extractable plant oils.

Preferably, compositions according to the present invention comprise combinations of essential oil and encapsulated essential oil mixtures in ratios of, for example, 50 50, 75:25 and 25 75, or ratios therebetween Preferably, the combinations are based upon the weight of the product The fragment of fungal cell may comprise a fungal cell wall, such as a ghost cell, or a part thereof wherein said part is capable of passively retaining the composition Encapsulated compounds are described In WO 00/69440.

The composition preferably contains an essential oil, essential oil mixture and/or a biocide as an active ingredient In an amount effective to inhibit the growth of a pathogen on a surface to which the composition is applied The active Ingredient is preferably present in the composition In an amount such that when the composition is applied to a surface, the active Ingredient is preferably present in an amount of from about 5 to about 30,ug/cm2 on or over said surface.

The fungal cell or fungal cell fragment Is preferably derivable from flamentous fungi, more preferably from fungi having relatively high levels of chitin and/or chtosan. Such species include, but are not limited to, Mucor and Rhzomucor, although other species that are lowemn chain, such as Pencllum, Apergllus and Fusarum may also be suitable The fungal cell or fungal cell fragment may be derived from Saccharomyces cerev/siae, such as Bakers yeast, Williams yeast (obtainable from Aventne Renewable Energy Co., Inc 1300 South 2nd Street, Pekin, Illinois, 61555- 00, USA) or DCL blue label yeast obtainable from Lessafre at www. lesaffreyeastcorp.com.

The fungal cell or fungal cell fragment may be derived from yeast that Is grown continually or grown In a batch. Yeast grown continually Is usually used for the production of ethanol for fuel purposes and is adapted to a high alcohol environment Such yeast Is termed ethanol yeast or bofuel yeast of which Williams yeast Is an example. Most preferably the fungal cell or fungal cell fragment Is derived from bofuel yeast.

The composition may be applied to the epithelum exposed by a wound or of a mucous membrane.

The mucous membrane may be the membrane lining the oral cavity or buccal cavity, tongue, stomach, small intestine (duodenum or jejunum), large intestine (colon), rectum, vagina, cervix, nose, naso- pharynx, or pulmonary system (trachea, larynx, bronchi, and lungs). The mucous membrane may be the membrane lining of the dgeshve system of humans, domestic pets, and livestock.

The mucous membrane may be the lining of the oral cavity, buccal cavity or the tongue where the composition of the present invention may be for pharmaceutical use, oral health care, or as an over the counter (OTC) medicine. To deliver to the mouth or tongue, the composition of the present nvenbon may be formulated as a powder, gel, spray, or tablet to treat for example, mouth ulcers, trench mount, gingvibs or canker sores. Compositions of the present Invention may be formulated In the form of a dry or liquid (emulsion or suspension) syrup, a sachet, a chewable, a chewing gum, an orodspersible, a dispersible effervescent, a dispersible tablet, a compressed buccal tablet, a compressed sublingual tablet, a chewable tablet, and a lozenge. Chewable dosage forms for drug delivery are well known to the pharmaceutical industry.

The mucous membrane may be the membrane lining the pharynx/throat where the encapsulated product can be for pharmaceutical use or as an OTC medicine. The composition of the present Invention may be formulated as a compressed sweet or boiled sweet To deliver the composition to the nasopharyngeal membranes, the composbon of the present Invention may be formulated as a powder, gel, spray or aerosol.

The mucous membrane may be the membrane lining the oesophagus or stomach, where the composition of the present invention can be for pharmaceutical use, nutnceutcal applicators, or as an OTC medicine. The composition of the present invention can be Incorporated In a one- or two-part gelatin capsule or other similar material to aid swallowing and prevent premature release of the active in the mouth or on the surface of the tongue.

The mucous membrane may be the membrane lining the colon/rectum where the composition of the present invention may be for pharmaceutical use, or as an OTC medicine. Delivery of actves to the colon or rectum can also be achieved through the use composition of the present Invention formulated as a suppository, ointment, cream, or gel.

The mucous membrane may be the membrane lining the nose, where the composition of the present invention may be for pharmaceutical use or as an OTC medicine. The composition of the present invention may be delivered as snuff, or as an aerosol delivered to the nose via nasal applicators as an aid for introducing the compositions of the present invention Into thenasopharyngeal space of a patient.

The mucous membrane may be the membrane lining the pulmonary system (i.e larynx, trachea, bronchi, and lungs) The mucous membrane may be the membrane lining the vagna/cervix where the composition of the present nvenbon may be for pharmaceutical use or as an OTC medicine. The composition of the present Invention may be formulated as a pessary, cream, ointment or gel. The mucous membrane may be the membrane lining the dgesbve system of humans, domestic pets, and livestock For example, delayed and controlled release of an active can take place when the active is released throughout the entire digestive system of humans. The biological membrane In this Instance Is the tunica mucosa, which lines the upper gastrointestinal tract, stomach, small Intestine and colon.

Accordingly, the composition of the present nvenbon may be formulated as a dry or liquid (emulsion or suspension) syrup, a sachet, a chewable, a chewing gum, an orodispersible, a dispersible effervescent, a dispersible tablet, a compressed buccal tablet, a compressed sublingual tablet, a chewable tablet, a melt-in-the-mouth, a lozenge, a paste, a powder, a gel, a tablet, a compressed sweet, a boiled sweet, a cream, a suppository, a snuff, a spray, an aerosol, a pessary, or an ointment.

In accordance with a further aspect of the present invention there Is provided a method of manufacturing a composition as described hereinabove comprising contacting a capsule with the composition such that the composition Is encapsulated by the capsule and retained passively In accordance with a further aspect of the present Invention, there is provided a wound dressing composing a carrier and a composition as described hereinabove.

The carrier may comprise any material capable of being sanitized eg. By radiation, and non-irritating to damaged tissue. Preferably, the Gamer comprises a non-woven synthetic material. More preferably, the Gamer comprises polyester.

The carrier may be formed into steps or patches Strips or patches may be layered to promote retention of the composition between layers.

The composition may be disposed on said carrier or the carrier may be impregnated with said composition.

The wound dressing may be packaged to prevent contaminator and/or damage whilst in storage or transit In one embodiment, the packaged wound dressing comprises a heat sealable envelope sealed around its perimeter with a wound dressing according to the present invention disposed therein.

The present invention further provides a method of producing an encapsulated material comprising treating a grown intact microbe such as a fungus or bacterium by contiguous contact with an encapsulatable material In liquid form The encapsulatable material being capable of diffusing into the microbial cell without causing total Iysabon thereof, and said treatment being carried out In the absence or presence of an organic lipd-extendng substance (as defined in European Patent Specification No. 0085805) as solvent or mcrodspersant for the encapsulatable material and in the absence of a plasmolyser, whereby the material is absorbed by the microbe by diffusion across the microbial cell wall and is retained passively within the microbe (as described in European Patent Specification 0085805). The aforementioned prior methods rely either on special microbe cultivation conditions to enhance the microbial lipid content to a very high level or on the use of a lipid- extending substance, and the materials to be encapsulated must be either soluble in the microbial lipid or soluble or mcrodispersible in the lipid- extending substance, respectively.

In French Patent Specification No. 2179528 there is described a method of causing certain materials to be absorbed and/or axed by microbes, In which a microbe such as pressed industrial yeast is treated with a plasmolyser, i.e a substance which causes contraction or shrinking of the microbial cytoplasm by exosmoss of cytoplasmic fluid, and then an aqueous solution of a material such as neodymium chloride, magnesium chloride or onion juice is added under certain conditions so that the aqueous material Is absorbed In place of the extracted cytoplasmic fluid In one embodiment, the fungal cell is In grown form, eat has been harvested from its culture medium, and is intact, i.e not Iysed Suitably the microbe Is alive, at least at the commencement of the treatment, however, a microbe which has been subjected to conditions (such as by irradiation of the microbe) to destroy its ability of propagate may be employed.

Preferably the capsule has a large size (cell size), for example of average diameter more than about 5 microns. Bactena may have a smaller normal cell size of about 1 to 2 microns but may be cultivated to attain a larger size.

It is not necessary for the capsule to have a significant lipid content. Typically the lipid content may be not more than about 5%, for instance up to 3%, by dry weight of the microbe.

The encapsulatable material should be in liquid form during the treatment. It may be a liquid in its normal state, or it may be normally a solid In which case it should be dissolved or mcrodispersed in a solvent that is not miscible with the microbial lipid. Examples of suitable solvents are the lower alcohols such as methanol, ethanol and so-propanol. The solvent may be removed after the encapsulation treatment, such as by spray-drying In one embodiment, the composition further comprises a carrier for coencapsulation with biocide or essential oil.

In one embodiment, where the composition comprises an essential oil and a biocidal compound, the \ carrier comprises the essential oil.

The encapsulatable material need not be soluble in any lipid forming part of the capsule The method of encapsulation preferably comprises mixing the capsule with the composition In a squid medium, especially an aqueous medium, to attain good dispersion and contact of the capsule with the composition. Accordingly, the composition may be mixed with an aqueous paste or slurry of the capsule, or the composition in a small quantity of water may be mixed with dry microbe. Preferably the composition forms an emulsion In the aqueous medium.

Encapsulation may be performed at normal ambient temperatures but preferably the temperature is elevated, at least during the Initial stages, such as during at least the first 30 minutes, In order to expedite the encapsulation. A suitable elevated temperature may be In the range 35 to 70 C, more preferably 45-60 C.

The encapsulation may be observed microscopically as one or more globules of the composition Inside the capsule. This may take a few hours.

In one embodiment, the capsule may be pretreated at an elevated temperature and/or with a proteolybc enzyme and/or with a chemical such as sodium hydroxide or a magnesium salt to enhance permeability prior to or in some cases during the encapsulation process. Such pretreatment may be carried out by Incubating the microbe In water at an elevated temperature The microbe may then be mixed with the material to be encapsulated at a lower temperature.

After encapsulation, the capsule may be treated to soften it in order to facilitate subsequent release of the encapsulated material, such as by treatment with a proteolybc enzyme or an alkali, or it may be treated to harden it in order to prevent premature liberation of the encapsulated material, such as by treatment with a dilute aqueous aldehyde solution The encapsulated material may be released from the capsules when desired by, for instance, chemical, bodegradabon or mechanical rupture of the microbial cell wall, and/or by subjecting the capsules to an environment In which the material diffuses gradually out through pores in the capsule and/or contacting the fungal cell wall or fragment thereof with epithelial cells and/or contacting the capsules with a compound that breaks down or disrupts the structure of cell membrane.

Capsules produced by the Invention give rise to controlled release characteristics; for example when the release of the encapsulated material is delayed or prolonged by a slow or gradual rupture of the capsule or slow diffusion therefrom providing a sustained treatment.

Specific embodiments of the present invention will now be described, by way of example only, with reference to the following examples 1.0 Selected staphylococcal strains Strains of staphylococcus (n=36) are as follows S aureus (n=2), S. epidermidis (n=2), S. I saprophybcus (n=1), S. haemolyticus (n=1), Methcllin sensitive S. aureus (MSSA) (n=6), Methclln resistant S. aureus (MRSA) (n=9), Epidemic methcilln resistant S. aureus (EMRSA) (n=15). Within the EMRSA strains selected, 3 strains are currently causing major problems within hospitals internationally 1.1, Selected essential oils Manuka oil (leptospermum Scoparium), Geranium (Egypt) (Pelargonium graveolens), Lavender Eastern Europe (Lavandula angustifolia), Lemongrass (East Indian) (Cymbopogon flexuosus), Tea tree (Melaleuca altern/folia).

1 1.1 Preparation of encapsulated essential oils Individual essential oils were encapsulated In washed Williams yeast. This was achieved by combining essential oil, washed Williams yeast and tap water in ratios of 1.2:4. The three components were mixed for 4 hours at 40 C and then spray dried at 170 - 180 C. This resulted In a fine powder of encapsulated essential oils. The volume of essential oil retained within the yeast was determined by gas chromatography (pI/mg).

1 1.2 Preparation of essential oil and encapsulated essential oil combnabons Combinations of essential oil and encapsulated essential oil mixtures were performed in ratios of 50.50, 75:25 and 25:75. To obtain an equivalent of the essential oil combinations in the encapsulated product, the combinations were carried out based upon the weight of the product.

The combnabons were as follows: Manuka + Geranium/ Geranium + Lavender/ Manuka + Lavender/ Geranium + Lemongrass/ Manuka + Lemongrass/ Geranium + Tea tree/ Manuka + Tea tree/ Lavender + Lemongrass/ Lemongrass + Tea tree/ Lavender + Tea tree Example of essential oil combinations: 75. 25 Lemongrass and Lavender - 1600,ul of essential oil (12001J1 Lemongrass: 400 pI Lavender) + 400,ul of MB = 2 ml of 80 % combined oil - 1 ml of 80 % used to create dilutions and 1 ml added to 19 ml of STA = 4 % oil Example of encapsulated essential oil combinations: 75 25 Manuka and Tea tree - 2286 mg of Manuka (750,ul oil) + 957 mg of Tea tree (250 pl oil) added to 9 ml of MB =10mlof10%o'1 1 ml c, 1C 0/c t=zl Ga3 c1 cil,.; +n:l, ,,i --1 1! r Ir, r,-, c:-T, = r, , 2 Es,,-n,'con o, t,4 t;/7nirr'ii,-'i,3i'iorJ c',-ic=-lt:,-,- ''lAI';,, o ll s::,--- a:,si sngI:,-, combnecl essental ois (Drec7 coniaci) Each nclivdual essental oil and essenial ol combnahon (see sechon 1 12) was clluted from 80r0/u to! 0 63% usng antboDc assay broth (AAB) Each dluton (1 ml) was then vortex mixecl wth 15 ml of molten senstvty test agar (STA) ancl dspensed nto ndvdual petn clshes Plates were allowecl to set then dried for 30 minutes After acidbon of the dlutons to the STA, each essental ol or essental ol combnaton resulted n a dlution of 4% to 0.031% An overnght broth culhre (ONBC) of each bactenal strain was diuted 1/10 usng AA6 Each stran was then placed onto the surface of the STA contarnng the essental ol and essential orl combnatrons, using a mulb-pont inoculator Plates were dned for 20 mnutes and then ncubated for 24 hours at 37 C The MIC of each stran was determned as the frst plate wthin the diluton senes ë showng no growth of the organsm ë - oeee

RESULTS .e

Tbe MIC of single essential oils anainst_II strains of staphylococu. I .Essenbal oils strainLavender Geranium Lemongrass ManukaTea tree OXford S aureus NCTC 65711% 2% 0.5% 0125%_0125% a aureus NCBC 11832 1 % 1 % _ 1 0% _ 0125% 0250% S. epidermidis NCTC 1 1 047 1 % 1 % 05% 0.125% 0250% S epidermidis NCTC 7944 1 % 1 % _ 1 0% 0.5% 0250% S saprophybcL's NCIMB 8711 _ 1% 1% 1.0% 0125% 0125% S haemolyticus NCTC 1 1 042 1 % 1 % 1. 0% 0125% 0125% Strain T1 MSSA 1% 1% 1.0% 0.125% 0125% Strain T4 MSSA 1% 1% 1 0% 0125% 0.062% MSSA (4) 1 % 2% 1 0% 0125% 0125% SSA (46) 1% 1% 1;0% 0.125% 0250% vlSSA (47) 1% 1% 1.0% 0.125% 0125% MSSA (48) 1% 2% 1.0% 0.125% 0.125% MRSA 11 1 % 2% 1 0% 0.5% 0062% MRSA 12 1% 1% 1. 0% 0.125% 0062%MRSA 13 1 % 1 % 1.0% 0.25% 0125% MRSA 14 1% ! 1% _1 0% 0125% 0125% MRSA 15 1% 1 2% 1.0% 025% 0125% la.

MRSA 16 _1 1% 1 2% !0 5% _ 1 0 125% 1 0 125% MRSA 17 _ _ _1%_ 1 2% 11 0% _ 0 125% 0 125% MRSA20, _1% 1 2% 1 1 0 /0 0 125% 0 125% MRSA 26 _ _1 10/0 1 _ 1 0% 0 125% 0 250% EMRSA m97 271 031 phage group 1 1% 1_ 2% 1 Q%0 125% 0 250% EMRSA m97 271 038 phage group 2 _ 1% 1% _ 1 0%_ 0 125% 0 125% EMRSAJ95 922 phage group 3 1% 1% i 0%_ 0 125% 0.125% EMRSA m97271052 phage group 4 _ 1% 2% _1.0%0.125%_ 0 125% EMRSA m972 71041 phage group 5 1% 2% 0.5% 0 125% 0 062% EMRSA m97 271 088 phage group 6_1% 2% 1 0 /0 0 125% 0 250% -'MRSA m97 271 047 phage group 81% 2% 1 0% = 0.125% 0 125% _MRSA m972 710 40 phage group 91% 1% _ 1 0%_ 0.125% _ 0 062% _MRSA m97 271 032 phage group 101% _ 1% 1.0% O 125% 0 125% EMRSA m97 271 036 phage group 111% _ 1% _ 1 0% 0.i25% 0.062% EMRSA m97 271 042 phage group 121% 1% 1 0% _ 0 125% 0 250% EMRSA m97 271 064 phage group 141% 1% _ 1.0% 0 125% 0 062% EMRSA g96 139 515 phage group 15 _1% 1% 1. 0%_ O 5% 0.062% EMRSA g96 138 744 phage group 16 1% 2% 0.5% 0 125% 0.125% EMRSA g96 136 210 phage group 17 1% 1% _ 1 0% 0 125% 0.062% The standard deviation for each was zero .e- . .e .. . ë ..* . . . . ..

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IN BRIEF

The MIC of essential oils and essential oil combinations that gave full inhibition of all strains of StaDhVloCOCCi.

IEssenbal oil MIC (%) l Lavender 1 Geranium 2 l Lemongrass 1 Manuka 0.50 Tea tree 0 25 Essential oil combinations 50:50 (MIC 75:25 (MIC 25:75 (MIC %) %) %) Manuka and Geranium 0.5 0.031 0.5 Manuka and Tea tree 0.125 0 031 (0.5) 2 Manuka and Lemongrass 0.5 0.031 0.5 Manuka and Lavender 0.5 0 25 1 Tea tree and Lemongrass 0.125 1 1 Tea tree and Lavender 0.125 1 Tea tree and Geranium 0 5 Lemongrass and Lavender 0 5 0.125 0 5 (1*) Lemongrass and Geranium 0.125 (0.25*) 0 5 0 5 Geranium and Lavender 0.5 1 * This MIC is for one strain only

CONCLUSION

Essential oils and essential oil combinations that gave the best results were: % Manuka and 25 % Geranium % Manuka and 25 % Tea tree 75% Manuka and 25 % Lemongrass Combinations of some essential oils were more effective than when the Individual essential oils were used singly.

3 Estimation of the Minimum inhibitory concentration (MIC) of staphylococci against combined encapsulated essential oils (direct contact).

The encapsulated essential oil combinations are as described hereinabove. Each encapsulated essential oil combnabon was diluted from 80% to 0.63% using antibiotic assay broth (MB). Each dilution (1 ml) was then vortex mixed with 19 ml of molten sensitivity test agar (STA) and dispensed Into individual petri dishes. Plates were allowed to set than dried for 30 minutes. After addition of the dilutions to the STA, each encapsulated essential oil combination resulted in a dilution of 4% to 0 031 %.

An overnight broth culture (ONBC) of each bacterial strain was diluted 1/10 using MB. Each strain was then placed onto the surface of the STA containing the encapsulated essential oil combinations, using a multipont Inoculator Plates were dried for 20 minutes then incubated for 24 hours at 37 C The MIC of each strain was determined as the first plate within the dilution series showing no growth of the organism

RESULTS

The MIC of encapsulated essential on combinations against all staphviococcal strains

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i Esssntial ol cornbnaL:ons SLrans I Manuka and geran Im 75 25 Manuka and tea tree 75 25 Manul<a and lemon rass 75 25 1 2Average 1 2 Aveage 1 2 A/eraJe Oxford S aureus NCTC _. ._ 65-/1 0 063% 0 063%0 063% 0 125% 0 25% 0 25% 0 063% 0 125% 0 125% S aureus NCBC 11862 _ 0 063% 0 063%0 063% 0 125% 0 250/0 0 25% 0 063% 0 125% 0 125% 3 epderrnids NCTC 11047 _ 1 0 063% 0 063% 0 063% 0 25% 0 25% 0 250/u 0 125% 0 125% 0 125% 3 epdermidis NCTC 7944 0 063% 0 063% 0 063% 0 25% 0 25% 0 25% 0 125% 0 125% 0 125% saprophytcus NCIMB 3711 _- 0 063% 0 063% 0 063% 0 25% 0 25% 0 25% 0 063% 0 1 25% 0 125% i haemolytcus NCTC M042 _0 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 063% 0 125% 0 125% itran T1 MSSA_ 0 063% 0 063% 0 063% 0 25% 0 25% 0 25% 0 125% 0 125% 0 125% itram T4 MSSA0 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 125% 0 125% 0 125% 1 dSSA (4)_ 0 063% 0 125% 0 125% 0 25% 0 125% 0 25% 0.063% 0 063% 0 063% 1 vlSSA (46)0 063% 0 063% 0 063% 0 125% 0 125% 0 125% 0 125% 0 125% 0 125% r \ASSA t47)0.063% 0 125 X, O 125% 0 125% 0.125% 0 125% 0 063% 0.063% 0. 063% i v1SSA (48)_ 0 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 125% 0 1 25% 0 125% \dRSA 110 063% 0 063% 0 063% 0 25% 0 125% 0 25% 0 125% 0 1 25% 0 1 25% dRSA 12_ 0 063% 0 063% 0 063% 0 125% 0 125% 0.125% 0 125% 0 125% 0 125% 1 vIRSA 130 063% 0 063% 0.063% 0 125% 0 125% 0 125% 0 125% 0 125% 0.125% . . v7RSA 140 063% 0 063% 0 063% 0 125% 0 125% 0 125% 0 125% 0 1 25% 0 1 25% VIRSA 150 063% 0 063% 0 063% 0 25% 0 25% 0 25% 0 125% 0 125% 0 125% 1 /IRSA 160.063% 0 125% 0 125% 0 125% 0 125% 0 125% 0 010% 0 032% 0 032% \4RSA 170 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 125% 0 125% 0 125% viRSA 200 063% 0 063% 0 063% 0 125% 0 125% 0 125% 0 063% 0 063% 0.063% -MRSA m97 271 031 P0 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 125% 0 125% 0 125% . 3rp 1 0 063% 0 125%0 125% 0.125% 0 125% 0 125% 0.063% 0 1 25% 0 125% 1 MRSA m97 271 038 P rp 2 0 063% 0 063%0 063% 0 25% 0 125% 0 125% 0 063% 0 125% 0 125% -MRSA J95 922 P grp 3 0 063% 0 063%0 063% 0 25% 0 25% 0 25% 0 125 / 0.1 25% 0 1 25% 1 MRSA m97271052 P grp 0 MRSA m972 71041 P 0 063% 0 063%0 063% 0.125% 0 125% 0 125% 0 125% 0 125% 0.125% - . rp 50 063% 0 063% 0 063% 0 125% 0 125% 0 125% 0 125% 0.1 25% 0 1 25% 1 MRSA m97 271 088 grp 6 0 063% 0 063%0 063% 0 125% 0 125% 0 125% 0 063 / 0 063% 0 063% MRSA m97 271 047 O -MRSA m972 710 40 0 063% 0 063%0 063% 0 125% 0 25% 0 25% 0 063% 0 125% 0 125% D grp 9 0.063% 0 125%0 125% 0 125% 0 25% 0 25% 0 063 % 0.1 2 5% 0 1 25% EMRSA m97 271 032 p grp 10 0 063% 0 063%0 063% 0 125% 0 25% 0 25% 0 125% 0 1 25% 0 125% EMRSA m97 271 036 Pgrp 11 0063% 0063%0063% 0 125% 025% 025% 0 125 / 0 125% 0 125% EMRSA m97 271 042 u P.grp 12 _ 0 063% 0 063%0 063% 0 125% 0 25% 0 25% 0 125% 0 125% 0 125% EMRSA m97 271 064 P grp 14 0 063% 0 063%0 063% 0 125% 0 25% 0 25% 0 125% 0 1 25% 0 1 25% EMRSA 996 139 515 P gr 1 _ - 0 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 125 / 0 125% 0 125% EMRSA 996 138 744 P gr I O 16 0 063% 0 063% O rJfi3% 0 125% 0 25% 0 25% 0 125% 0.1 25% 0 1 25% EMRSA 996 136 210 P gr 1 1 17 0 063% 0 063% 0 063% 0 125% 0 25% 0 25% 0 125% 0 1 25% 0.1 25% i The highest MIC of differing MlC's was used as an average. A difference in MlC's may have been due to experimental error.

CONCLUSION

All encapsulated combnabons were effective at inhibiting growth of all strains of staphylococci.

Combinations of some encapsulated essential oils were more effective than when using the individual encapsulated essential oils alone.

Combined encapsulated essential oils had a higher MIC than the combined non- encapsulated essential oils However, the encapsulated products may be retaining the oils within the yeast cells and potentially prolonging release 4. Assessment of the vapours of single and combined essential oils against strains of staphylococci (Vapour phase) Neat (100%) individual essential oils and essential oil combinations (see section 1.1.2) (201) were placed onto individual 6 mm filter paper disks and the discs placed onto the lids of Individual petri dishes An overnight broth culture (ONBC) of each bacterial strain was diluted 1/100 using MB and swabbed over the surface of a STA plate Plates were dried for 20 minutes. The disks were then placed Into the petri dish lid and the lid placed onto the petri dish. All plates were Incubated for 24 hours at 37 C. The ZOI of each strain was determined by measuring the area of bacterial clearing (diameter, mm).

10 strains were initially used to screen If the essential oils or combinations were effective If shown to be effective the oil was assessed against all strains.

RESULTS

(1) The ZOI of 10 strains of staPhYiococcus tested acainst the vaPours of single essential oils Essential oils Strain G=m | Manuka J Oxford S. aureus NCTC 6571 FG FG FG S. epidermids NCTC 11047 FG FG FG S. haemolyticus NCTC 11042 FG FG FG Strain T4 MSSA FG FG FG MRSA 12 FG FG FG MRSA 13 FG FG FG MRSA 14 FG FG FG EMRSA m97 271 064 P.grp 14 FG FG FG EMRSA g96 139 515 P. grp 15 FG FG FG EMRSA 996 138 744 P. grp 16 FG FG FG FG=Full growth (no area of clearing) w The standard deviation of all was zero.

All three oils were not effectve so ther effect on all strains was not assessed.

(2) The ZOI of 10 strains of stanhviococcus anainst the vaPours of sinale essential oils; : Essen tial oils: Lemol [grass Tea tree Strains 1 2 Average SD+ 1 2 Average SD+ Oxford S aureus NCTC 6571 20 21 20.5 0 71 15 17 16 1 41 S. aureus NCBC 11882 25 24 24 5 0. 71 15 16 15 5 0.71 S. epidermidis NCTC 11047 85 87 86 1 41 FG FG FG FG S. epidermidis NCTC 7944 46 48 47 1.41 FG FG FG FG S. saprophybcus NCIMB 8711 42 45 43.5 2.12 FG FG FG FG S. haemolyticus NCTC 11042 20 16 18 2. 83 10 10 10 0.00 Strain T1 MSSA 30 31 30.5 0.71 FG FG FG FG Strain T4 MSSA 40 43 41.5 2.12 10 12 11 1 41 MSSA (4) 55 54 54.5 0 71 16 15 15.5 0 71 SSA (46) 1 27 25 1 261 1 41 i FG FG 1 FG FG = 3655354 1: 1 11 14 RSAl3: 3] 3 Z 12 i] as 2 [2 ilRSA 14 _ = U 35 31 2 12 1. I FG FG \/RSA 16 36 3g 37.5 2 12 23 25 24 1 4i ArsA 1 21_20 ZO 5 0 71 _ 25 29 272 83 2 21_ 19 20 1 41 _ 16 14 ___ 1 41 -MRSA rn97 271 031 phage group i |3026 _ 28 1 41 FG FG FG FG --MRSA m97 271 038 phage group 2 30 32 _ 31 1 41 FG FG_ FG FG MRSA j95 922 phage group 3 55 52 53 5 2 12 17 18 17 5 0 71 MRSA m97271052 phage group 4 65 61 63 2 83 25 26 25 5 0 71 MRSA m972 71041 phage group 5 50 49 _ 49 5 0 71 25 26 _ 25 5 0 71 MRSA m97 271 088 phage group 6 45 49 47 2 83 FG FG FG FG MRSA m97 271 047 phage group 8 27 23 25 2 83 12 10 11 1 4i -MRSA m972 710 40 phage group 9 50 42 46 5 66 10 11 10 5 0 71 _MRSA m97 271 032 phage group 10 29 26 27.5 2 12 15 17 16 1 41 _MRSA m97 271 036 phage group 11 47 46 46 5 0 71 11 _ 9 10 1 41 _MRSA m97 271 042 phage group 12 _ 43 4a 45 5 _ 3 54 21 25 23 2 83 . _MRSAm97 271064 phagegroup14 1 25 27 26 141 8 10 141 -MRSA g96 139 515 phage group 15 30 34 32 2.83 14 12 13 1 41 e -MRSA g96 138 744 phage group 16 _60 62 61 1 41 FG FG FG FG -MRSA g96 136 210 phage group 17 38 36 37 1 41 18 12 15 4 24 ë FG=Fuli growth (no area of cieanng) .-.e . (1) The ZOI of 10 strains of stanhviococcus against combined essential oils ë .- i . _ Essential oil combinations _ssential Oli 1 = 2 = 3 4 5 6 Strain * 1 2 Average SD+ * * * * Dxford S aureus NCTC 6571 FG 12 15 13.5 _ 2 121 FG FG FG FG S epidermidis NCTC 11047 FG FG FG _ FG FG FG FG FG FG _: S haemolyticus NCTC 11042 FG FG FG FG FG FG FG FG FG Strain T4 MSSA FG FG FG FG FG FG FG FG FG VIRSA 12 FG FG FG FG FG _ FG FG FG FG I _ SA13 | FG |FG 2U FG | FG | FG | FG | FG I FG i MRSA 14 | FG |FG FG| FG |_FG: FG FG | FG | FG EMRSA m97 271 064 phage group 14 l FG |FG FG FG FG I FG | FG I FG | FG EMRSAy96 139 515 phage group i5 1 FG l FG FG1 FG FG 1 FG EFG; FG FG EMRSA g96 13B 744 phaye group 16 7 FG FG FG I FG FG FG NG KEY essenbai ols _ % Manuka 50 % Geranum 1 % Tea tree 50 % Lavender 2 0 % Tea tree 50 % Geranum 3 % Manuka 50 % Tea tree 4 % Manuka 50 % Lavender 5 % Geranum 50 % Lavender 6

_

FG= FUJI growth (no area of cleanng) * SD s were for each was zero (2) The ZOI of 10 strains of staphviococcus against combined essential oils e : Essenbal oll combnatons e Essenbal oil Lemongrass and Lav nder Lemon! rass and 1 ea tree Stran 1 2 Mean SD+ 1 2 Mean SD+ Dxford S aureus NCTC 6571 _ 20 22 21 26 25 25 5 0 71 e S aureus NCBC 11882 20 22 21 1 41 12 11 11 5 0 71 epdermdsNCTC11047 30 34 32 2.83 46 49 475 212 epdermidis NCTC 7944 35 33 34 1.41 35 35 35 0.00 saprophybcus NCIMB 8711 _ 28 28 28 0 00 12 13 12 5 0 71 haemolyticus NCTC 11042 _ 30 33 315 2 12 35 33 34 1 41 train T1 MSSA _ 31 33 32 1 41 25 26 25.5 071 . ,train T4 MSSA 31 26 28.5 3 54 13 16 14 5 2 12 *... vlSSA (4) 35 36 35 5 0 71 42 45 43 5 2 12 vlSSA (46) 22 25 23 5 2 i2 15 21 18 4 24 vlSSA (47) _ 62 64 63 1 41 30 30 30 _ 0 00 vlSSA (48) 35 35 35 0 00 23 24 23 5 0 7i vlRSA11 15 16 155 071 12 14 13 141 vlRSA 12 20 18 19 1 41 15 18 165 2 12 vlRSA 13 17 18 17 5 0 71 20 23 215 2 12 sARSA14 5 6 55 071 15 15 15 000 RSA 15 31 33 32 1 41 16 18 _ 17 1 41 \1RSA 163_ 33 1 39 5! o 71 1 30 3" 1 31 1 1 '1 \AP5A 17 __j 22_0 _ 21 1 1 '1 1 25 _9 1 23 5 1 2 i2 _ \/IRSA 20 __1 26 22_ 21 1 15 14 | l4 5 1 _ 0 71 \/iRSA26 _ _ _ 32 30 31 _ 1 1 d 25 z2 5 1 2 12 _MRSA m97 271 031 phage group 1 5,7 _ 6 1.41 33 39 1 _ 33 5 1 _ 0 71 I MRSAm97 271 03;3 phage group2 45 47 46 1 4i 19 19 1 19 1 0 00 MRSAJ95 922 phaye group 3 30 31 30 5 0 71 49 a,2 1 42 _ 1 0 00 MRSAm97271052 phagegrou _ 30 25 27 5 3 54 40 40 1 40 _ 000 MRSA m972 71041 phage group 5 38 42 40 2 33 45 45 45 1 0 00 MRSA m97 271 088 phage yroup 6 0 33 _ 31 5 2 12 45 45 _ 45 _ 0 00 -MRSA m97 271 047 phage group 8 31 31 1 31 0 00 17 18 17 5 _ 0 71 -MRSA m972 710 40 phage group 9 24 25_ 24 5 0 71 32 33 32 5 0 71 MRSA m97 271 032 phage group 10 30 31 30 5 0 71 90 56 73 _ __ _ MRSA m97 271 036 phage group 11 25 27 i 26 1 4i 12 15 13 52 12 MRSA m97 271 042 phage group 12 20 19 1 19 5 0 71 25 24 24 50 71 1 MRSA m97 271 064 phage group 14 11 9 10 _1 41 10 9 _ 9 5_ 0 71 1 MRSA g96 139 515 phage group 15 20 23 1 21.5 2 12 18 18 180 00 1 -MRSA g9_p 15 18 _ 16 5 2 12 20 _ 20 20 0 00 -MRSA g96_136 210 phage group 17 40 40 40 _ 0 00 90 61 75 5 20 51 :- ë .e* ..- Essenbal ll comblnatlons * Essenbal oll _ _ Lemongrass and Manuka Lemongrass and Geranium e Strain_ _ 1 2 Mean SDr 12 Mean _ SD- .e Oxford S aureus NCTC 6571 _ _20 22 21 1 41 15 12 13 5 2 12 * S. aureus NCBC 11882 8 5 6 5 2 12 8 5 6 5 2 12 1 * S epidermidis NCTC 11047 42 45 _3 5 2 12 35 36 35 5 0 71 * S epidermidis NCTC 7944 48 48 48 0 00 29 30 29 5 0 71 S saprophybcus NCIMB 8711 34 38 36 2 83 19 18 18.5 0 71 S haemolybcus NCTC 11042 25 26 25.5 0 71 25 21 23 2 83 Strain T1 MSSA 10 11 10 5 0 71 10 10 10 0 00 Strain T4 MSSA 73 79 _6_ 4 24 15 14 14 5 0.71 MSSA (4) 32 33 32. 5 0 71 22 22 22 0 00 MSSA (46) 15 15 15 0 00 17 _18 17 5 0 71 MSSA (47) 32 32 32 0.00 21 21 21 0 00 MSSA (48) 19 18 18 5 0 71 15 14 14 5 0 71 /1RSt 11I FG FG FG I FG | 10 i l I 10 5 I u - _| vlPo. 12 _I 15 1-1 1 i37 2 3: 10 _ g _ 1 r 1 0 7 1 1 \17RSA 13 __ = = = r l / _ I'- I_ I,l _ O rr _1 \ARSA i4 1 22 2' 1 23 1 141 I 15 16 1 155 L 07i iARSA -i5 _ _ L 15 16 1 i5 5 1 0 71 _ 19 17 1 13: 1 41 \7RSA 16 ___ 1 20 =r20 5 0 71 _ _ 29 2g-1 29 1 0 00 1 ll7RSA 17 _1 30 30 1 30 0 00_ _ 9 9 1 g 1 0 00 \/IRSA20 21 2 1 22 5 2 12 16 13 1 17 1 41 JIRSA 26 = 25 24 24 5 0 71 25 _ 26 1 25 5 0 71 -MRSA m97 27 i 03 i phage group 1 52 55 53 5 2 12 3838 38 _ 0 00 MRSA m97 271 038 phage group 2 17 17 17 0 00 j19 19 19 _ 0 00 MRSA j95 922 phage group 3 23 23 23 0 00 4247 44 5 _ 3 54 I MRSA m97271052 phage group 4 25 25 25 0 00 140 _ 42 41 1 41 I MRSA m972 71041 phage group 5 37 37 37 0 00 |45 44 44 5 0 71 22 21 _ 0 71 145 44 44 5 _ 0 71 j -MRSA m97 271 047 phage group 8 26 29 27 5 2 12 j17 17 _ 17 _ 0 00 MRSA m972 710 40 phage group 9 1 25 24 24 5 0 71 3232 32 0 00 --MRSA m97 271 032 phage group 10 _ 35 34 34 5 0 71 1 14 15 14 5 0 71 MRSA m97 271 036 phage group 11_ 38_ 36 37 1 41 23_ 21 _ 22 _1 41 MRSA m97 271 042 phage group 12 28 29 28 5 0 71 _ 25 24 24 5 0 71 _MRSA m97 271 064 phage group 14 13 13 13 0 00 18 18 18 0 00 -MRSA g96 139 515 phage group 15 11 10 10 5 0 71 18 _ 18 18 0 00 -MRSA g96 138 744 phage group 16 30 26 28 2 83 30 31 _30 5 0 71 _MRSA g96 136 210 phage group 17 30 32 31 1 41 25 29 27 2 83 -ee e - ZOI for essential orls and essential orl combnabons ananst all strans Essenbal ol Cleanng (range mm) e - Lavender FG _ Geranum FG . _ Lemongrass 18-86 Manuka FG

_ _

Tea tree FG-25 5 FG = Full growth (no area of cleanng) * = Intal tests dd not show good results so the effect of the ols on all strans was not pursued 50:50 ZOI 75 25 ZOI 25:75 ZOI (range, Essential oil combination (range, mm) (range, mm) mm) Manuka and Geranium FG* FG* FG* Manuka and Tea tree FG* FG* FG-31.5 Manuka and Lemongrass FG-76 FG* FG-72.5 Manuka and Lavender FG* FG* FG* Tea tree and Lemongrass 9.5-75.5 FG-41 FG-60.5 Tea tree and Lavender FG* FG* FG * Tea tree and Geranium FG* FG-24 5 FG* Lemongrass and Lavender 5.5-63 11.5-53.5 FG-52.5 Lemongrass and Geranium 6.5-44.5 126 5 FG* Geranium and Lavender FG* FG* FG* FG = Full growth (no area of clearing) * = Initial tests did not show good results so the effect of the oils on all strains was not pursued

CONCLUSION

The vapours of essential oils and essential oil combinations that gave the best results (greatest ZOI) were.

% Lemongrass and 50 % Tea tree % Lemongrass and 25 % Lavender 75% Lemongrass and 25 % Geranium All three combinations were used In further studies The vapours of combinations of some essential oils were more effective than when the essential oils were used singly.

5. Estimation of the Minimum inhibitory concentrations (MIC) of all strains against Triclosan (Direct contact) A stock solution of triclosan was prepared by adding 256 mg of triclosan to 10 ml of Dimethyl sulphoxide. A working solution was then prepared by diluting 1 ml of the stock solution In 9 ml of antibiotic assay broth (MB). The working solution was then diluted from 2560 fig ml to 0.31,g ml using MB. Each dilution of tnclosan (1 ml) was then vortex mixed with 19 ml of molten sensitivity test agar (STA) and poured into petri dishes. When set, the dilutions of triclosan in the STA ranged from 1281lg ml to 0.011lg ml.

An overnight broth culture (ONBC) of each bacterial strain was diluted 1/100 using MB. Each strain was then placed onto the surface of the plates using a mult'-pont Inoculator. Each plate was dried for 20 minutes and incubated for 24 hours at 37 C The MIC of each strain was determined as the first plate in the dilution series showing no growth of the organism.

RESULTS

The MIC for triclosan against all strains of staphylococci Strain MIC (Mg ml' ) Oxford S aureus NCTC 6571 0.63 S aureus NCBC 11882 2 S epidermidis NCTC 11047 0.63 S. epidermids NCTC 7944 2 S. saprophyticus NCIMB 8711 2 S haemolytcus NCTC 11042 1 Strain T1 MSSA 0.5 Strain T4 MSSA 0 25 MSSA (4) 0 5 MSSA (46) 2 MSSA (47) 0.5 MSSA (48) 1 MRSA 11 0.5 MRSA 12 0 5 MRSA 13 2 MRSA 14 0.5 MRSA 15 1 MRSA 16 0.062 MRSA 17 0.25 MRSA 20 1 MRSA 26 2 EMRSA m97 271 031 phage group 1 0.5, EMRSA m97 271 038 phage group 2 0. 5 l EMRSA j95 922 phage group 3 0 5 EMRSA m97271052 phage group 4 0.062 EMRSA m972 71041 phage group 5 0.062 EMRSA m97 271 088 phage group 6 1 EMRSA m97 271 047 phage group 8 2 EMRSA m972 710 40 phage group 9 1 EMRSA m97 271 032 phage group 10 0.5 EMRSA m97 271 036 phage group 11 0.5 EMRSA m97 271 042 phage group 12 1 EMRSA m97 271 064 phage group 14 0 5 _MRSA 996 139 515 phage group 15 2 EMRSA 996 138 744 phage group 16 1 EMRSA 996 136 210 phage group 17 1 I The standard deviation for all was zero.

CONCLUSION

The concentration of triclosan able to inhibit all strains of staphylococci was 2 p9 my 6. Estmabon of the Minimum inhibitory concentration (MIC) of combined triclosan and essential oils against all staphylococci strains (Direct contact) Tnclosan (4 p9 ml) was added to 0.063 % essential oil in equal volumes (concentrations of each were determined in previous experiments). This resulted in a 1/2 dilution of each component (2 9 ml tnclosan and 0. 031 % essential oil) The combination was then double diluted and 1 ml of each dilution added to 19 ml of molten STA and dispensed Into individual petri dishes. Each was allowed to set and dried for 30 minutes.

An overnight broth culture of each bacterial strain was then diluted 1/10 using MB Each strain was then placed onto the surface of the STA containing each combination of oil and triclosan using An overntght broth culture of each bacienal siran was then diluiSc 1/10 usng B Each strarn WES then placecl onio the sur,ace of lhe STA contanny each combnaton of ol and tnclosan usng a rnult-potnt Inocuiator Each plal:e was allowed to ciry for 20 mnutes and ncubated for 24 hours at 37 G T he MIC ol each strain was cletermined as the frst plate within Lhe cllution senes contanng no growth of the organisra

RESULTS

The MIC of combined essential oils and tnclosa I aaainst all stra ls of staphviococcl Manoka and Manuka and Manuka and Tea tree and Lemongrass and Stran._ Geranum (1) Tea tree (2) Lemongrass (3) Lavender (4) Geranum (5) xford S aureus NCTC 6571 F __ E _ F D aureus NCBC 11882 _ _ D _ D_ ___ i. epidermds NCTC 11047_ = _ E _ E - _ __ E epdermids NCTC 7944 _ F _ _ D __ _ DD ___ saprophytcus NCIMB 8711 _ F D _ _ D _ _ >. haemolybcus NCTC 11042 _ F _ D F __ D _E itran T1 MSSA _ _ _F _F E _ D E train T4 MSSA_ _ F _ E_ _ = E _ _ _ e -VISSA (4)_ F E _ D _ E dSSA (46) F D D | F D v1SSA (47) _ F F _ F D_ _ e I vlRSA 11 F I E_ E_ D _ E vlRSA 12 _ F _ E _ E L) E e /IRSA 13 F _ _D D F C 41RSA 14 _ F F E D E vlRSA 15 _ _ _ F I F __ E D E dRSA 16 _ F _ F E D _ .e vlRSA 17 _ F F D D _ E e (ARSA 20 F F F _ F _ E | -MRSA m97 271 031 phage F _ D _ D= _ D E 3roup 1 _ F _ F E F E MRSA m97 271 038 phage _ _ 3roup 2 F _ E F D _ E -MRSA j95 922 phage group 3 F F F F E MRSA m97271052 phage _ _ _ _ __ rouP 4 F F E F MRSA m972 71041 phage _ _ _ _ _ _ E __ 3roup 5 F F F F E MRSA m97 271 088 phage _ F _ _F_ E _ F _ E hIR5,'- m97 2, l 0,6 pl-lage groul3 1_ 1 1 I E MR5A mr3/ 27 1 542 phage yroup I -- 1 _ _ 12 _ _ I F E E F F -i\/lPoA m97 27 i 064 phage group. ._ _ _ __ - 14. _ _ _ F E F E MRSA 996 139 5 15 phage group _ MRSAg96 13372-!phaye group I- F - E I E F _ D __ _MRSA g96 136 210 phage group _ __ __ ___ 17 _ F E _ E F | E

-

--- 29 3 Key for oils 4 and 5 _ Oli = Trciosan _ Oli = Tnclosan A 0 031 + 2 A 0 125, 2 B 0016 - 1 B 0063 __ _ 1 C 0008 _. _ 0.5 C 0031 + 05 D 0 004 +_0 25 D 0 016 - 0 25 E 0 002 +0 125 E 0 008 + 0 125 F _ 0 001 _ +0 0625_ F 0.004 0 0625 Q 0 0005_ + 0 03125 G 0 002 _+ 0 03125

CONCLUSION

A lower concentration of combned tnciosan and essential ols were more effectve at ë.

nhbiDng growth of all strains compared to when used singly 7 Assessment of the vapours of toclosan against all strains of staphylococcus (Vapour phase) -e a A stock solution of triclosan was prepared by addng 256 mg of trclosan to 10 ml of D'methyl sulphoxde A worictng solution was then prepared by dlubng 1 ml of the stock soluton in 9 ml of anDbotc assay broth (MB) The working solution (20,ul) was then placed onto 6 mm filter paper b dscs and placed nto the ltd of petn dishes An ONBC of each bactenal stran was dluted 1/100 and swabbed onto the surface of STA The lids contaning the discs were placed onto the petn dshes and the plates ncubated for 24 hours at 37 =C The ZOI of each stran was determned by measuring the area of bactenal cleanag (dameter, mm)

RESULTS

The ZOI of all strains anainst tnclosan vapours Strain 1 2 Average | SD+ Oxford S. aureus NCTC 6571 50 52 51 1 41 S. aureus NCBC 11882 47 49 48 1 41 S. epdermidis NCTC 11047 50 51 50.5 0 71 S. epdermidis NCTC 7944 50 51 50 5 0.71 S. saprophyticus NCIMB 8711 33 42 37 5 6.36 S haemolyticus NCTC 11042 48 44 46 2.83 Stran T1 MSSA 46 45 45.5 0.71 Strain T4 MSSA 50 52 51 1.41 MSSA (4) 50 52 51 1.41 MSSA (46j 45 45 45 0 00 MSSA (47) 52 51 51.5 0.71 MSSA (48) 49 48 48.5 0 71 MRSA 11 43 43 43 0 00 MRSA 12 45 44 44.5 0 71 MRSA 13 47 47 47 0 00 ! MRSA 14 55 55 55 0.00 MRSA 15 50 50 50 0.00 MRSA 16 55 56 55.5 0 71 MRSA 17 31 33 32 1.41 -1 MRSA 20 55 55 55 0 00 MRSA 26 _ 40 40 40 0 00, EMRSA m97 271 031 phage group 1 42 40 41 1.41; EMRSA m97 271 038 phage group 2 57 57 57 0 00 EMRSA j95 922 phage group 3 55 53 54 1.41 1 EMRSA m97271052 phage group 4 50 50 50 0 EMRSA m972 71041 phage group 5 55 55 55 O EMRSA m97 271 088 phage group 6 52 52 52 0 EMRSA m97 271 047 phage group 8 52 52 52 EMRSA m972 710 40 phage group 9 55 55 55 EMRSA m97 271 032 phage group 10 49 49 49 0 EMRSA m97 271 036 phage group 11 52 52 52 0 EMRSA m97 271 042 phage group 12 45 45 45 O EMRSA m97 271 064 phage group 14 35 35 35 0 EMRSA g96 139 515 phage group 15 47 47 47 0 EMRSA g96 138 744 phage group 16 50 50 50 0 EMRSA g96 136 210 phage group 17 47 47 47 0 SD for alNs zero

CONCLUSION

The vapours of toclosan were effective at tnhibiDny growth of all staphylococcal stratus 8 Assessment of the vapours of tnclosan and essential oils against all strains of staphylococcus (Vapour phase) Tnclosan (2560pg mid) was added in equal volumes to essential oil (100%) (The concentrations of essential oils were determined In previous experiments) This resulted In a 1/2 dilution of each component (1280rg ml tnclosan and 50% essential oil) The combination was then added to a 6 mm filter paper disc and placed on the lid of a petri dish An ONBC of each bacterial strain was diluted 1/100 and swabbed onto the surface of STA The lids containing the discs were placed onto the petri dishes and the plates Incubated for 24 hours at 37

TIC

The STA of the petri dishes was then surface swabbed with a 1/100 dilution of the ONBC of 10 selected staphylococci strains and the petn dish placed onto the petri dish Plates were Incubated a...

for 24 hours at 37 C The ZOI of each strain was determined by measuring the area of bacterial . clearing (diameter, mm) a. , Note 10 strains were Initially screened to assess If they had any effect on growth

RESULTS

b The ZOI of 10 staphviococcal strains against the vapours of combined tnclosan and essential oils a-e. Tea tree and Lemongrass and Lemongrass and Strain Lemongrass Lavender Geranium Oxford S aureus NCTC 6571 FG 19 _ FG S aureus NCBC 11882 30 FG FG__ epidermdis NCTC 11047 35 FG FG S haemolybcus NCTC 11042 FG FG FG Strain T4 MSSA 15 FG MRSA 12 12 FG 20 MRSA 13 29 FG 10_ MRS.'- 12! | Fry I FC, I FC- I EMRSAn1C727I061LnaSeJIO!IP14 j FO i FG | _ FG I l EFARSA 996 1 39] F- I FG -I EMRSA g96 1 3S 7'!4 Pl1a9= 9rOLIP 16 | FG | FG; FG FG= FLIII growil1 (10 area of cleanny) Stanclarcl devEtbons for each were zero

CONCLUSION

c When essential oils and tnclosan were combined, the effect of the vapours on the ZOI was less effective than when used singly This Indicates that when combined an antagonishc effect between the tnclosan and oils were occurring 9 Assessment of essential oil and encapsulated essential oil combinations on the growth of EMRSA 16 when Incorporated Into calcium alpinate for use as a wound dressing Essenbal oil combinations (100 pI, combinations previously determined) were added to 20 ml of calcium, alginate and then placed into the lid of a petri dish An ONBC of EMRSA 16 was diluted 1/100 and swabbed onto the surface of STA The lid was placed on the petri dish and the plates Incubated at 37 C for 24 hours RESULTS ë ZOI of EMRSA 16 against combinations of essential and encapsulated essential oils In calcium alqinate I - - _ Essenbal oil Encapsulated essential oil | Sea tree and Lemongrass 50 50 No growth No growth _ Lemongrass and Lavender 75 25 No growth No growth |Lemongrass andgeranium 75 25_ No growth No growth

CONCLUSION

Both the essential oils and encapsulated essential oils prohibited growth of strain EMRSA over 24 hours.

Assessment of essential oil and encapsulated essential oil combinations on the growth of EMRSA 16 when Incorporated Into gauze for use as a wound dressing Essential oil combinations (100,ul, combinations previously determined) were added to 10 ml of water and then used to soak a 15cm2 piece of added to gauze An ONBC of EMRSA 16 was diluted 1/100 and swabbed onto the surface of STA The gauze dressing was then placed over the top of the petri dish and the lid replaced (the gauze and the STA were not touching). The plates were then incubated for 24 hours at 37 C and the ZOI measured

RESULTS

ZOI of EMRSA 16 against combinations of essential and encapsulated essential oils contained in gauze Essential oil Encapsulated essential oil Tea tree and Lemongrass 50:50 No growth No growth Lemongrass and Lavender 75:25 No growth No growth Lemongrass and geranium 75.25 No growth No growth

CONCLUSION

References 1. Lawrence JO. Burn Bacteriology during the last 50 years. Burns 1992; 18: (suppl 2), 23-29 2 Chlds, C., Edwards-Jones, V, Heathcote, D M, Dawson, M. & Davenport, P Patterns of Staphylococcus aureus colonization, toxin production, Immunity and illness In burned children Bruns (1994) 20, 514-521.

3. Voss A. M'latovic D. Wallrauch-Schwarz C Rosdahl VT Braveny I, Methiclln-Resstant Staphylococcus Aureus In Europe Eur J Clan Microbiol Infect Dis 1994, 13. 50-55 4 Walker J. Borrow R. Edwards-Jones V, Oppenheim BA, Fox AJ. Epdemological characterization of methicillin-resistant Staphylococcus aureus isolated In the North West of England by protein A (spa) and coagulase (coa) gene polymorphisms. Epdemiol Infect.

1998. 121: 507-514.

5. Anon., 1998 Methicillin-resistant Staogtk/ciccys ayreys, Commun D's Rep Weekly. 8 372.

6 Smith TL Pearson MC, Wilcox R Cruz C. Lancaster MV. Robinson-Dunn B. Tenover FC.

Zervos MJ B and JD White E. Jarvis JO. Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-lntermediate Staphlococcus aureus Working Group.

New. Engl. Med. 1999, 240: 493-501.

7. Lawrence JC Dressings and wound Infection. America Journal of Surgery 1994; 167: (suppl 1 A), 21 S-24S.

8. Edwards-Jones, V. Dawson, MM, & Childs, C A survey into TSS in UK Burns Units Burns, 2000.

9 Herruzo-Cabrera, R. Vizcaino-Alcaide, M.J. Mayer, R. F. & Rey-Calero, J. A new in vitro model to test the effectiveness of topical antimicrobial agents. Use of an artificial eschar Burns, 1992; 18; 35-38.

Claims

1 A composition for use as a medicament wherein the composition comprises at least one essential oil and a fungal cell or fungal cell fragment wherein molecules of the at least one essential oil are encapsulated or partially encapsulated by the fungal cell or fungal cell fragment.

2. A composition compnsng at least one essential oil and at least one biocidal compound.

3 A composition as claimed in claim 2 wherein the bocdal compound comprises a fungicide, an antibiotic and/or a bactericide

4 A composition as claimed In claim 3 wherein the bocdal compound comprises one or more selected from the group comprising tnclosan, mupirocin, chlorhexidine, povidone Iodine and silver sulphadiazine.

5. A composition comprising an essential oil, a biocdal compound and a fungal cell or fungal cell fragment, wherein molecules of at least one of the essential oil or biocdal compound are encapsulated or partially encapsulated by the fungal cell or fungal cell fragment

6. A composition comprising two or more essential oils and a fungal cell or fungal cell fragment wherein molecules of at least one essential Dims encapsulated or partially encapsulated by the fungal cell or fungal cell fragment.

7. A therapeutic formulation composing a composition as claimed In any one of the previous claims.

8 The use of a composition for the manufacture of a medicament for the treatment of mcrobaMnfection, the composition comprising at least one essential oil and a fungal cell or fungal cell fragment, wherein molecules of the essential oil are encapsulated or partially encapsulated by the fungal cell or fungal cell fragment

9 The use of a composition as claimed in claim 8 wherein the use Is for the treatment of Staphylococcus Infection.

The use of a composition as claimed In claim 9 wherein the use is for the treatment of S. aureus, S ep/dermidis, S. saprophyt/cus, S. haemolyticus, Methiclln sensitive S aureus (MSSA), Methcllin resistant S. aureus (MRSA) and/or Epidemic methcillin resistant S. aureus (EMRSA).

11. The use of a composition as claimed In claim 10 wherein the use Is for the treatment of

MRSA

12. A method of preventing a microbial infection In a subject comprising administering to a subject a composition as claimed in any one of claims 1 to 7.

13 A composition comprising a) a first essential oil composing Manuka and a second essential oil comprising one or more essential oil selected from the group composing Geranium, Lavender, Lemongrass, and Tea tree; or b) a first essential oil comprising Geranium and a second essential oil comprising one or more essential oil selected from the group compnsng Manuka, Lavender, Lemongrass, and Tea tree; or c) a first essential oil comprising Lemongrass and a second essential oil composing one or more essential oil selected from the group composing Geranium, Lavender, Manuka, and Tea tree; or d) a first essential oil composing Lavender and a second essential oil composing one or more essential oil selected from the group composing Geranium, Manuka, Lemongrass, and Tea tree; or e) a first essential oil composing Tea tree and a second essential oil comprising one or more essential oil selected from the group comprising Geranium, Lavender, Lemongrass, and Manuka.

14 A wound dressing comprising a carrier and a composbon or formulation as claimed in any one of claims 1 - 7 and

13.