EP1089754A2 - Agent antimicrobien efficace contre les elements pathogenes gram positif - Google Patents

Agent antimicrobien efficace contre les elements pathogenes gram positif

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Publication number
EP1089754A2
EP1089754A2 EP99928186A EP99928186A EP1089754A2 EP 1089754 A2 EP1089754 A2 EP 1089754A2 EP 99928186 A EP99928186 A EP 99928186A EP 99928186 A EP99928186 A EP 99928186A EP 1089754 A2 EP1089754 A2 EP 1089754A2
Authority
EP
European Patent Office
Prior art keywords
lacticin
quarters
resistant
teat
gram
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99928186A
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German (de)
English (en)
Inventor
Reynolds Paul Ross
Colin Hill
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University College Cork
Teagasc Agriculture and Food Development Authority
Original Assignee
University College Cork
Teagasc Agriculture and Food Development Authority
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University College Cork, Teagasc Agriculture and Food Development Authority filed Critical University College Cork
Publication of EP1089754A2 publication Critical patent/EP1089754A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0041—Mammary glands, e.g. breasts, udder; Intramammary administration

Definitions

  • the present invention relates to an antimicrobial agent which is effective against gram-positive human pathogens.
  • the invention provides the use of the bacteriocin lacticin 3147 in the preparation of a medicament for the treatment of infections caused by Gram +ve organisms, and to pharmaceutical compositions containing lacticin 3147.
  • Bacteriocins are antimicrobial peptides which can have a broad spectrum of inhibition. Some undergo substantial post-translational modifications, e.g. nisin which is a 34 amino acid peptide containing a number of unusual amino acids, including dehydrated residues and five cross-linking lanthionine residues (Jack et al., 1995). This bacteriocin belongs to a class of bacteriocins termed lantibiotics, to reflect both the presence of these unusual residues and its broad host range antimicrobial activity.
  • Lacticin 3147 is produced by the GRAS (generally regarded as safe) organism Lactococcus lactis DPC3147 (Ryan et al., 1996). The genetic determinants which encode lacticin 3147 are encoded on a 60.2 kb plasmid, pMRCOl . Lacticin 3147 is the subject of PCT Patent Application No. PCT/IE96/00022, published as WO 96/32482.
  • Lacticin 3147 exhibits a wide host range, though confined to Gram positive organisms, including foodborne pathogens such as Clostridium botulinum, Listeria monocytogenes, Bacillus subtilis and Bacillus cereus.
  • An important distinction between nisin and lacticin 3147 is that the latter is effective at physiological pH.
  • nisin displays poor solubility (and thus poor activity) at pH 7, which has to date limited its clinical applications in the treatment of human disease.
  • lacticin 3147 is a membrane-active pore-forming complex, which exhibits a bactericidal mode of action, causing cell death but not cell lysis. Energised cells are more sensitive, suggesting that the presence of a proton motive force (PMF) promotes the interaction of the bacteriocin with the cytoplasmic membrane, leading to the formation of pores.
  • the pores are selective for ions, in particular potassium and inorganic phosphate.
  • MRSAs methicillin-resistant Staphlococcus aureus
  • Lacticin 3147 was isolated from an organism found in milk and mastitis affects the milk-producing gland. One might expect a milk-borne agent to effect an organism found in milk or related to milk production. One could not predict with certainty that the agent would be an effective against non-milk-related organisms.
  • lacticin 3147 was investigated for its ability to inhibit a number of Gram positive pathogens, including two methicillin-resistant S. aureus (MRSA) isolates, vancomycin-resistant Enterococcus (VRE), penicillin-resistant Pneumococcus (PRP), Propionibacterium acne and Streptococcus mutatis.
  • MRSA methicillin-resistant S. aureus
  • VRE vancomycin-resistant Enterococcus
  • PRP penicillin-resistant Pneumococcus
  • Propionibacterium acne Propionibacterium acne
  • Streptococcus mutatis The pathogens were chosen to represent organisms with differing sites of infection, including respiratory, meningial, skin, oral, wound and cardiac; as well as including some of the more problematic antibiotic-resistant strains.
  • lacticin 3147 in the manufacture of a medicament for the treatment or prevention of infections caused by Gram-positive bacteria.
  • the bacteria are multiplely drug-resistant organisms and in particular antibody-resistant bacteria.
  • the Gram-positive bacteria are selected from Enterococcus, Staphylococcus aureus, Pneumococcus, Propionibacterium acne, Streptococcus mutans, Listeria monocytogenes, Clostridium perfringens, and Cloistridium pere.
  • the bacteria may be human pathogens.
  • compositions for the treatment or prevention of infections caused by Gram-positive bacteria comprising lacticin 3147.
  • Pharmaceutical compositions may further comprise a lacticin 3147 induced-bacteriocidal-enhancing amount of glucose.
  • the pharmaceutical composition may be adapted for topical, parenteral, oral, sub-cutaneous or intra-venous application.
  • the composition may be a selected from mouth wash, a toothpaste, a topical skin preparation including those used for acne treatment, an antiseptic soap, an inhaler, an intra-venous application, an oral ingestion preparation or an antiseptic wipe or the like.
  • the pharmaceutical composition may further comprise a lacticin 3147 induced- bacteriocidal-enhancing amount of glucose.
  • the invention provides a method of treatment of the human or animal body by the application of lacticin 3147 for the treatment or prevention of bacterial infection, particularly infection by Gram +ve organisms.
  • the method of treatment may also comprise use of a lacticin 3147 induced-bacteriocidal-enhancing amount of glucose.
  • Figure 1 Inhibitory action of lacticin 3147 against the gram-positive species L. lactis subsp. cremoris HP (A), MRSA 13 (B), MRSA 148 (C), penicillin-resistant Pneumococcus (D), vancomycin-resistant Enterococcus (E), P. acne (F), and 5. mutans (G) illustrating the difference in sensitivity of the test strains.
  • FIG. 1 Bactericidal effect of lacticin 3147 on the viability of: (A) MRSA 13, (B) MRSA 148, (C) penicillin resistant Pneumococcus, (D) vancomycin-resistant Enterococcus, (E) Propionibacterium acne, and (F) Streptococcus mutans. ( ⁇ ) no addition, ( ⁇ ) addition of lacticin 3147 at a concentration of 20,000 AU/ml. Data points along the horizontal axis represent 0% survival. Data points represent the average of experiments performed in duplicate.
  • Figure 4 Recovery of deliberately infected 5 aureus 5246 infused at a dose of 1,500- 1,800 cfu to quarters of lactating cows containing either teat seal only or a teat seal/lacticin 3147 formulation.
  • Bacterial strains and culture conditions The bacteriocin producer L. lactis subsp. lactis DPC3147 was grown at 30°C in M17 (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 0.5% (w/v) glucose, as was the standard indicator strain L. lactis subsp. cremoris HP.
  • Pathogenic strains included: vancomycin resistant Enterococcus (VRE) (Beaumont Hospital, Dublin, Ireland), grown in Tryptone Soy Broth (TSB) (Difco Laboratories, Detroit, USA) supplemented with 0.6% (wt/vol) yeast extract (Oxoid); Methicillin resistant Staphylococcus aureus (MRSA) 13 ⁇ 148 (Mercy Hospital, Cork, Ireland), grown in Brain Heart Infusion (BHI) broth (Oxoid); Penicillin-resistant Pneumococcus (PRP) 856 (Mercy Hospital), grown in BHI broth; Propionibacterium acne (ATCC6919 American Type Culture Collection, Maryland, USA), grown in Reinforced Clostridial broth (RCB) (Oxoid); and Streptococcus mutans 257 (Professor W. Bowen, University of Rochester, New York, U.S.A.) grown in TSB. All pathogens were grown at 37°C without aeration.
  • lacticin 3147 used for these studies was prepared as follows; TY broth ( ⁇ -glycerophosphate at 19 g/L, glucose at 10 g/L, yeast extract at 5 g/L, tryptone at 2.5 g/L, MgSO 4 7H 2 0) at 0.25 g/L, MnSO 4 4H 2 O at 0.05 g/L, pH 6.75) was cleared of contaminating proteins which bind to hydrophobic binding XAD-16 beads (Sigma) by passing it through 50 g of beads. Lactococcus lactis
  • DPC3147 was then propagated in this TY broth overnight at 30°C.
  • the cells were removed by centrifugation at 12,000 rpm.
  • the bacteriocin-containing supernatant was incubated with 25 g XAD-16 beads with agitation for 30 min, at which point a further 25g XAD-16 beads were added, and incubation with agitation was allowed to proceed for a further 30 min, allowing the bacteriocin to bind.
  • the beads were then washed with distilled water, followed by washes with 40% ethanol, and the bacteriocin was subsequently eluted with 70% isopropanol, lOmM acetic acid, pH 2.
  • Bacteriocin activity was determined by the agar well diffusion assay, as described by Ryan et al, 1996. Molten agar at 48°C was seeded with the indicator strain L. lactis subsp. cremoris HP (50 ml of an overnight culture per 20 ml agar), dispensed into sterile petri-dishes, and allowed to solidify. Wells of approximately 4.6 mm in diameter were made, and 50 ml aliquots of a two-fold serial dilution of the bacteriocin preparation were dispensed into the wells. After overnight incubation at 30°C, bacteriocin activity was calculated as the inverse of the last dilution that gave a definite zone of clearance after overnight incubation. Activity units were expressed per millilitre (1/dilution x 20).
  • lacticin 3147 The bactericidal effect of lacticin 3147 on the six pathogens was investigated by two methods.
  • A Initially agar well diffusion assays were performed. These were carried out as described above, except that the agar was seeded with the pathogenic strain.
  • B Time-kill curve studies were then performed on the pathogens. Sensitive cells were inoculated at 10% (from an overnight culture) and grown to mid-exponential phase. The cells were washed, resuspended and diluted in 2.5 mM sodium phosphate buffer, pH 7, supplemented with 10 mM glucose, so that upon addition of bacteriocin at 20,000 AU/ml the bacterial count was 10 to 10 6 cfu. An equal number of cells in buffer without bacteriocin was used as a control. Samples were then taken at appropriate intervals over a 2 hour period to determine the viable cell count.
  • teat sealTM An internal intramammary teat sealer similar in composition to a commercial teat seal product currently on the market (Teat sealTM; Cross Vetpharm Group, Ltd., Dublin) was used for this study.
  • a teat seal comprises a heavy inorganic salt in a paraffin/wax base which forms a plug in the teat sinus and acts as a physical barrier to infection.
  • Teat seals containing lacticin 3147 were prepared as described by Ryan et al (1998) except the teat seal was blended with 1% (wt/wt) Tween 80 and left overnight prior to the addition of a lacticin 3147 concentrate (lOO ⁇ l/gram of teat seal/Tween80). The blended formulation was filled into a sterile 4 ml syringe and stored at 4°C until use.
  • S. aureus DPC5246 was used as the challenge organism to test the efficacy of lacticin 3147 in vivo.
  • One bead of the stock culture was removed and streaked over the surface of an ABA plate and incubated overnight at 37°C.
  • Individual colonies were subcultured from the plate into 10 ml of BHI broth and incubated for 6 h at 37°C.
  • the number of viable cells was counted and then diluted to the required number of colony forming units (cfu) per ml in 10% sterile antibiotic-free skim milk.
  • the diluted culture was stored in 10 ml aliquots at -20°C until required.
  • each teat Prior to infusion of the teat seals, the tip of each teat was disinfected with a cotton wool swab soaked in methylated spirits. After morning milking, 2 teats in each cow were infused with teat seal plus lacticin 3147 formulation (4g) and the 2 remaining teats in each cow were used as untreated controls. The teat seal was not manipulated in the teat after infusion, so as to allow it to form a plug in the teat sinus and duct. Two hours later all the teats were inoculated to a depth of 17 mm with the S. aureus DPC5246 challenge inoculum using a syringe with a blunted smoothed tip to prevent injury to the teat. Cows were not milked again until the next morning (approximately 18 h later). Teat seals were then removed from each udder quarter, foremilk samples were taken in an aseptic manner from all challenged quarters and the microbiological status assessed.
  • Milk or dry-period secretions from individual udder quarters were microbiologically assessed by streaking out a loopful (approx. 10 ⁇ l) on separate quandrants on the surface of ABA plates and incubating aerobically for 16 h at 37°C.
  • Streptococcus dysgalactiae M was selected as the challenge organism because previous in vitro studies showed that lacticin 3147 was effective against this pathogen.
  • This isolate classified as Strep, dysgalactiae spp. dysgalactiae by SDS-PAGE total protein profiling (BCCMTM Culture Collection; Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) was previously recovered from a case of clinical mastitis and had been preserved in a microbiological bead storage system (Protect; bacterial preservers; Technical Service Consultants Ltd., Lancashire, England) prior to the start of this study.
  • ABA aesculin blood agar plate
  • the ABA was prepared from blood agar base #2 (Lab M, Bury, England) to which 0.1% aesculin and 7% citrated whole calf blood were added.
  • Two hundred fifty millilitres of brain heart infusion broth (Oxoid Ltd., Hampshire, England) were inoculated with the Strep, dysgalactiae M culture and incubated at 37°C for 8 h.
  • a total bacterial count was carried out on the 8-h stock, which was then diluted to produce a working concentration of 1.5 x 10 cfu/ml in 10% sterile antibiotic-free skim milk.
  • a 0.1-ml aliquot of skim milk containing 1.5 x 10 cfu of streptococci was inoculated into treated and control udder quarters via the teat canal and deposited into the teat sinus at a depth of 17 mm from the tip of the teat. This experimental technique was used primarily to demonstrate the combined effect of the lacticin 3147 with the protection afforded by the teat seal alone.
  • Animal Trial 2 In a further trial, 16 cows were selected. Based on SSC data and microbiological analyses, 59 quarters were deemed suitable for the trial. From these, 29 quarters were infused with the lacticin 3147 plus teat seal formulation containing 32,768 AU of bacteriocin, one was infused with teat seal containing only 1% Tween 80 and the remaining 29 quarters served as untreated controls. All 59 quarters were infected with 0.1 ml of S. aureus DPC5246 using an inoculum level of 1,500-1,800 cfu/0.1 ml. The S. aureus challenge survived in 18 of the 29 control quarters (62.1%; Table 1). In contrast, the S.
  • aureus challenge was recovered from only 4 of the 29 teats (14% recovery) in quarters containing the teat seal plus lacticin 3147 formulation.
  • the S. aureus challenge survived in the quarter containing the mixture of teat seal and 1% Tween 80.
  • S. aureus isolates in the foremilk samples were enumerated to assess if a difference in the recoveries between treatments had occurred. With the exception of one quarter, the number of viable S. aureus recovered from the treated quarters was noticeably reduced relative to the untreated quarters (Fig. 4).
  • Overall the presence of the teat seal plus lacticin 3147 formulation significantly reduced (P ⁇ 0.001) the recoveries of S. aureus.
  • RAPD PCR profiles for 14 of the Strep, dysgalactiae isolates recovered from the quarters treated with seal and 2 of the Strep, dysgalactiae isolates recovered from quarters treated with seal plus lacticin 3147 confirmed that the infections were caused by the challenge strain.
  • lacticin 3147 The problem of antibiotic resistance can be addressed from two very different angles with the use of lacticin 3147. As discussed above, it has potential in the treatment of antibiotic resistant infections. But it may also have a role to play in reducing the occurrence of antibiotic resistance. The potential of lacticin 3147 in the prevention of mastitis has previously been demonstrated (Ryan et al, 1998). The overuse of antibiotics in veterinary medicine is thought to be a major contributing factor to the prevalence of antibiotic-resistant bacteria. Replacing antibiotics with lacticin 3147 in the treatment/prevention of mastitis would be a step towards a reduction in antibiotic use.
  • lacticin 3147 inhibits the dominant Gram-positive etiological agents of bovine mastitis namely, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalacatiae, Streptococcus bovis, and Streptococcus uberis.
  • the efficacy of a combination of lacticin 3147 and an intramammary dry cow teat seal was assessed in animals by deliberately challenging teats of non-lactating cows with Strep, dysgalactiae. The level of challenge was deliberately set to cause approximately a 50% failure rate of the teat seal alone.
  • Teat seal plus lacticin 3147 was significantly more successful in preventing infection (6% of quarters infected) compared to quarters containing teat seal alone (61% infected). The ability of teat seal plus lacticin 3147 to inhibit Staph. aureus was also assessed in teats of lactating cows. In one trial, the teat seal plus lacticin 3147 reduced the incidence of teats shedding Staph. aureus to 14%, compared to 66% in untreated quarters. These studies highlight the potential of lacticin 3147 as a non-antibiotic food grade anti-microbial suitable for the prevention of bovine mastitis. Lacticin 3147 also inhibits many medically important human pathogens including methicillin-resistant Staph.
  • lacticin 3147 may find uses in human therapy and may provide an attractive addition to the range of anti-microbials available to inhibit antibiotic resistant Gram-positive organisms.
  • Lacticin 3147 a broad-spectrum bacteriocin which selectively dissipates the membrane potential. Appl. Environ. Microbiol. 64:439-445.
  • lTwo of the infections were caused by Staphylococcus aureus.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention se rapporte à un agent antimicrobien qui est doté d'une efficacité dirigée contre une plage d'organismes Gram positif. Ledit agent antimicrobien est la bactériocine Lacticine 3147, qui s'avère efficace à la fois in vivo et in vitro contre une plage d'organismes Gram positifs.
EP99928186A 1998-06-22 1999-06-22 Agent antimicrobien efficace contre les elements pathogenes gram positif Withdrawn EP1089754A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IE980499 1998-06-22
IE980499 1998-06-22
PCT/IE1999/000057 WO1999066949A2 (fr) 1998-06-22 1999-06-22 Agent antimicrobien efficace contre les elements pathogenes gram positif

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EP1089754A2 true EP1089754A2 (fr) 2001-04-11

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EP (1) EP1089754A2 (fr)
AU (1) AU4529699A (fr)
WO (1) WO1999066949A2 (fr)

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WO2009003040A1 (fr) 2007-06-27 2008-12-31 Jcr Technologies Llc Procédé de stérilisation haute pression à l'état congelé
EP2227484A1 (fr) * 2007-11-30 2010-09-15 TEAGASC, The Agriculture and Food Development Authority Thuricine cd, un antimicrobien pour cibler spécifiquement clostridium difficile
DK3053446T3 (da) 2014-12-19 2019-05-27 Csk Food Enrichment Bv Fremgangsmåde til fremstilling af schweizerost
EA035622B1 (ru) 2014-12-19 2020-07-16 СиЭсКей ФУД ЭНРИЧМЕНТ Б.В. Способ производства сыра типа швейцарского

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IES70514B2 (en) * 1995-04-12 1996-12-11 Teagasc Agric Food Dev Authori Bacteriocins

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Publication number Publication date
AU4529699A (en) 2000-01-10
WO1999066949A2 (fr) 1999-12-29
WO1999066949A3 (fr) 2000-02-24

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