Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/10—Animals; Substances produced thereby or obtained therefrom
  • A01N63/14—Insects
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/56—Materials from animals other than mammals
  • A61K35/63—Arthropods
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
  • A01N61/02—Mineral oils; Tar oils; Tar; Distillates, extracts or conversion products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/65—Tetracyclines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

• the present invention relates to a method of treating a surface populated by bacteria capable of producing a biofilm using secretions of Lucilia sericata larvae and to compositions useful in such a method.
• Biofilms are biological films which develop and persist at surfaces. They may be found on the surfaces of industrial equipment where liquids are transported or processed, in plumbing systems, and on surfaces adjacent to such equipment or systems. They are often found on the surfaces of medical implants or devices inserted into the body. They may also form in areas of the body which are open to the air; in particular they may be found in wounds and on the lining of the lungs.
• a biofilm can be described as a bacterial population enclosed within a polysaccharide matrix which adheres to surfaces.
• Biofilms are generally stable formations which are difficult to treat by conventional techniques. This is due to the protective nature of a polysaccharide biofilm matrix in which the micro-organisms are embedded. Conventional medicaments, such as antibiotics, are less effective either due to diffusion barriers or the altered metabolic state of micro-organisms in the biofilm.
• Biofilm formation is thought to involve the production, by the microorganisms, of diffusible signal molecules by a process known as quorum sensing. These molecules are thought to trigger production, by the microorganisms, of exo-polysaccharides, exo-proteins and other secondary metabolites. Compounds which interfere with these molecular processes may inhibit biofilm formation and/or weaken an already formed biofilm.
• Pseudomonas aeruginosa is one of the most common and problematical of infective bacteria. It is particularly problematical in that it forms biofilms which are difficult to treat with conventional antibiotics. Biofilm formation by Pseudomonas aeruginosa is problematical for patients with cystic fibrosis in whom it colonises the lungs causing infections which are difficult to treat and often ultimately fatal. Efficient wound healing is a complex physiological process which involves many mechanisms including cell migration, growth factor secretion, angiogenesis, tissue n lelling and the intrinsic proteinase/antiproteinase balance of the wound contributing in concert and in an apparently staged manner to accelerate controlled tissue regeneration.
• Wound care products are essential in modern medical practice, especially for the treatment of patients with chronic wounds or burns.
• Many different substances have previously been proposed as having activities which contribute to the healing of wounds. These previously proposed substances include streptokinase, collagenase and streptodornase (all obtained from bacterial sources), bromelain (from pineapples), plasmin and trypsin (obtained from cattle) and krill enzymes (obtained from Crustacea).
• Clinical trial data indicate that such substances are only partially effective in promoting the healing of wounds.
• the larvae (maggots) of the green bottle fly, Lucilia sericata, are known to have significant wound healing attributes as live organisms. Debridement treatment using the larvae of Lucilia sericata has become a widely accepted clinical practice. However, little has been reported in the literature about the way in which these larvae go about their task of cleaning wounds to an extent that conventionally intransigent wounds heal. Healing can be mechanical, biochemical or a combination of both. Our work shows that the effects of these larvae can be mimicked using extra-corporeal secretions.
• live la ⁇ /ae are unpleasant to many patients and the use of live larvae on wounds and the introduction of their crude secretions into wounds, which inevitably occurs when the larvae are used, are unacceptable to many patients and to many medical practitioners.
• the use of live organisms also increases the risk of allergic reactions in the patient.
• the excretions/secretions (ES) of Lucilia sericata larvae are known to contain an enzyme which exhibits trypsin-like serine proteinase activity.
• This invention is based on the discovery that extra-corporeal ES also have the ability to break down the low molecular weight signalling molecules produced by bacteria to determine the density of the bacterial population and, thus, disrupt the bacterial messaging network on which biofilm formation depends.
• the present invt _ I provides, in a first aspect, a method of treating a surface populated by a bacteria capable of producing a biofilm which comprises contacting the surface with a substance having N-acyl homoserine lactone degradant activity obtained from the secretions/excretions of Lucilia sericata.
• the bacteria capable of producing a biofilm is Pseudomonas aeruginosa or Staphylococcus aureus.
• the healing of a chronic wound has been shown to be impaired by the presence of a bacterial infection.
• the level of infection affects the balance between healing and chronicity.
• the bacterial contribution to wound hypoxia and pathological effects are an impediment to efficient healing.
• the low molecular weight signalling molecules are known to include N-acyl homoserine lactones, e.g. N- butanoyl L-homoserine lactone (BHL) and N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) from .
• BHL butanoyl L-homoserine lactone
• OdDHL N-(3-oxododecanoyl)-L-homoserine lactone
• the secretions from Lucilia sericata larvae may be collected by washing sterile larvae with phosphate buffered saline followed by filtering, under sterile conditions.
• the present invention provides an antimicrobial composition comprising the secretions of Lucilia sericata larvae and one or more antibiotic compound.
• the antibiotic compound is tetracycline.
• the sterile secretions with or without the addition of a conventional antibiotic may be delivered onto a wound area using any known dermal delivery system or m ⁇ 3 incorporated into a sterile support, such as a poultice, to be applied to a wound area as a dressing.
• the collected secretions were assayed for protein content (BioRad protein assay) and protease activity (hydrolysis of fluorescein isothiocyanate labelled (FITC)-casein).
• the secretions were sterile filtered (22 ⁇ m filter) and aliquotted ready for use and stored at -20°C.
• the recovery of cells from cultures grown under biofilm producing conditions revealed differences when the cultures were grown in the presence of L sericata ES products.
• the culture was grown in 100 ⁇ l aliquots in a 96 well microtitre plate. This has the effect of increasing the surface area of liquid in contact with the plastic well surface in comparison with flask grown culture, thus promoting the growth of biofilm.
• P. aeruginosa was inoculated from an overnight culture and grown to early exponential phase before dilution (1/2000) to give ⁇ 10 3 cells per well. The culture was then grown in the presence of ES, inactivated ES (boiled 10min) or phosphate buffered saline (control).
• the culture was grown overnight at 37°C before collection together of each type of aliquot and centrifugation (13,000 x g for 10 min) to recover the cells.
• the addition of an aliquot of active ES to sample D (denatured ES) followed by incubation overnight at 37°C resulted in the removal of the slime layer originally formed.
• the slime layer may consist of exo-polysaccharide formed as part of the biofilm and removed by the action of glycosidase in L. sericata ES.
• the exo-polysaccharide has been suggested to be alginate (a polymer consisting of poly guluronic and mannuronic acids).
• thermostable PMSF/APMSF-sensitive activity from L. sericata Excretory/Secretory Products (ES)
• BHL and OdDHL may be quantified using thin layer chromatography (TLC) (RP18 F 2 5S or RP2 UV 254 plates respectively).
• TLC thin layer chromatography
• the particular organisms used emit light when in contact with BHL or HL. Therefore if the TLC plate is overlaid with soft agar containing the biosensor organism the position of the signalling molecule will be revealed by emission of light after a period of incubation.
• the intensity of the light emitted here is shown by converting to pseudo colour in which the most intense light shows as yellow with a gradation to the least intense - dark blue (Fig.7-side bar).
• Fig.7 demonstrate the effect of larval ES on degradation of BHL.
• the positive control (lane 5) showed light production from the BHL alone.
• This degradation was prevented by pre-incubation of the ES with phenylmethanesulphonyl fluoride (PMSF)(lane 4) and to a lesser extent 4- amidinophenyl-methanesulphonyl fluoride (APMSF)(lane 3)(inhibitors of serine protease activity).
• Boiling of the ES (lane 2) did not prevent degradation thus indicating thermal stability of the activity.
• Anti-microbial activity was assessed by the formation of bacteria-free plaques around wells containing 2 ⁇ l haemolymph in a bacterial lawn of E. coli D31.
• the wells (8) were formed in a regular pattern equidistant from the edge of the plate using a template.
• the anti-microbial activity was assessed by comparison with plaques produced by 2 ⁇ l Cecropin B (Sigma) at 100 ⁇ g/ml, 10 ⁇ g/ml, 1 ⁇ g/ml and 0.1 ⁇ g/ml (Fig.10).
• Haemolymph taken after 48h of induction by P. aeruginosa produced an antimicrobial plaque of 5mm diameter - greater than that produced by the 10 ⁇ g/ml cecropin standard (4.25mm) but smaller than the 100 ⁇ g/ml standard (8mm).

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Insects & Arthropods (AREA)
• Plant Pathology (AREA)
• Wood Science & Technology (AREA)
• General Chemical & Material Sciences (AREA)
• Pest Control & Pesticides (AREA)
• Agronomy & Crop Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Environmental Sciences (AREA)
• Dentistry (AREA)
• Epidemiology (AREA)
• Organic Chemistry (AREA)
• Virology (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Animal Husbandry (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Materials For Medical Uses (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

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• 2002
  • 2002-03-09 GB GBGB0205593.7A patent/GB0205593D0/en not_active Ceased
• 2003
  • 2003-03-06 EP EP03712317A patent/EP1485112A2/en not_active Withdrawn
  • 2003-03-06 WO PCT/GB2003/000959 patent/WO2003075654A2/en not_active Ceased
  • 2003-03-06 CA CA002478401A patent/CA2478401A1/en not_active Abandoned
  • 2003-03-06 JP JP2003573941A patent/JP2005525849A/ja active Pending
  • 2003-03-06 US US10/506,948 patent/US20050260183A1/en not_active Abandoned
  • 2003-03-06 CN CNB038099446A patent/CN100496514C/zh not_active Expired - Fee Related
  • 2003-03-06 GB GB0419331A patent/GB2401788B/en not_active Expired - Fee Related

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