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EP-3592155-B1 - METHOD FOR REDUCING CONTAMINATION OF AN OBJECT WITH CLOSTRIDIUM

EP3592155B1EP 3592155 B1EP3592155 B1EP 3592155B1EP-3592155-B1

Inventors

  • KAZANAVICIUTE, Vaiva
  • MISIUNAS, Audrius
  • RAZANSKIENE, Ausra

Dates

Publication Date
20260506
Application Date
20180307

Claims (14)

  1. A method of preventing or reducing contamination of food with Clostridium, comprising contacting said food with a composition comprising at least one endolysin active against said Clostridium, wherein at least one of said at least one endolysin is ZP173 comprising the amino acid sequence of SEQ ID NO: 2 or a derivative of this endolysin, and wherein said derivative or endolysin has a sequence identity of at least 90% to the amino acid sequence of SEQ ID NO: 2; or said derivative or endolysin has from 1 to 30 amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 2.
  2. The method according to claim 1, wherein said composition is or comprises a plant material or extract thereof, wherein the plant material is a material from a plant having expressed said at least one endolysin, preferably an edible plant having expressed said at least one endolysin.
  3. The method according to any one of claims 1 or 2, wherein said composition is an aqueous solution containing said at least one endolysin; or said composition is a lyophilized or dried powder containing said at least one endolysin, said powder may be dissolved or suspended in water or an aqueous solution before use.
  4. The method according to claim 3, wherein said aqueous solution containing said at least one endolysin is a buffered aqueous solution that contains from 50 to 400 mM NaCl, preferably from 140 to 310 mM, and may further contain a sulfhydryl compound.
  5. The method according to any one of claims 1 to 4, wherein said food is sprayed with an aqueous solution containing said at least one endolysin or is immersed into an aqueous solution containing said at least one endolysin.
  6. The method according to any one of claims 1 to 5, wherein said food is meat, preferably said meat is raw meat or cooked meat; or said food is gravy.
  7. The method according to any one of claims 1 to 6, wherein said Clostridium is Clostridium perfringens, preferably said Clostridium perfringens is anyone or more selected from of Type A, Type B, Type C, Type D and Type E, preferably a Type A strain containing enterotoxin CPE.
  8. The method according to claim 1, wherein said derivative or endolysin has a sequence identity of at least 95%, preferably at least 97%, to the amino acid sequence of SEQ ID NO: 2; or said derivative or endolysin has from 1 to 15, preferably from 1 to 10, amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 2.
  9. The method according to claim 1, wherein said composition is a buffered aqueous solution that contains from 150 to 700 mM NaCl, preferably from 200 to 550 mM NaCl, and/or the composition is an aqueous solution of a pH from 4 to 8, preferably from 4.5 to 7, more preferably from 5.0 to 6.5, and even more preferably from 5.0 to 6.0.
  10. The method according to claim 2, wherein said plant material is material from a plant selected from the group consisting of spinach, chard, beetroot, carrot, sugar beet, Nicotiana tabacum, Nicotiana benthamiana and/or said plant material is one or more leaves, roots, tubers, or seeds, or a crushed, milled or comminuted product of said leaves, roots, tubers, or seeds.
  11. A process of producing a composition comprising at least one endolysin active against Clostridium, said process comprising the following steps: (i) expressing said at least one endolysin in a plant, preferably an edible plant, (ii) harvesting plant material containing expressed endolysin from said plant, (iii) extracting said at least one endolysin from said plant material using an aqueous buffer to obtain a composition containing said at least one endolysin, (iv) optionally removing undesired contaminants from said composition, wherein at least one of said at least one endolysin is ZP173 comprising the amino acid sequence of SEQ ID NO: 2 or a derivative of this endolysin, wherein said derivative or endolysin has a sequence identity of at least 90% to the amino acid sequence of SEQ ID NO: 2; or said derivative or endolysin has from 1 to 30 amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 2.
  12. The process according to claim 11, wherein step (iv) comprises, in this order, a hydrophobic interaction chromatography step, a desalting step, and an ion exchange chromatography step.
  13. A composition comprising an endolysin active against Clostridium, wherein said endolysin is ZP173 comprising the amino acid sequence of SEQ ID NO: 2 or a derivative of this endolysin, wherein said derivative or endolysin has a sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 2; or said derivative or endolysin has from 1 to 30 amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 2.
  14. The composition according to claim 13 for use in a method of treating or preventing infection with Clostridium such as Clostridium difficile or Clostridium perfringens.

Description

FIELD OF THE INVENTION The present invention relates to method of preventing or reducing contamination of an object such as food or animal feed with Clostridium. The invention further relates to a process of producing a composition comprising at least one endolysin active against Clostridium, said process comprising expressing said endolysin in a plant or in plant cells. The invention further relates to a composition comprising an endolysin active against Clostridium, and to a method of treating an infection with Clostridium such as Clostridium difficile or Clostridium perfringens. BACKGROUND OF THE INVENTION The anaerobic spore-forming bacterium Clostridium perfringens is a source of one of the most common food-borne illnesses in the United States and Europe. C. perfringens gastroenteritis is caused by type A strains (producing alphatoxin encoded by the cpa gene, also known as phospholipase C, encoded by the plc gene) that produce the C. perfringens enterotoxin (CPE) (Xiao et al., 2012). The CPE-mediated food poisoning outbreaks typically involve a large number of victims and are associated with temperature-abused meat or poultry dishes. Optimal conditions for food poisonings arise when food contaminated with CPE-positive C. perfringens spores is slowly chilled or held or served at a temperature range of 10-54 °C, allowing germination and rapid growth of C. perfringens (Li and McClane, 2006, Lindström et al., 2011). The costs associated with the disease are high. In the Netherlands, the average incidence of Clostridium perfringens gastroenteritis is estimated to be 171,000 cases per year, with total cost from food-related C. perfringens infection cases amounting to 23.2 million euros (Mangen et al., 2015). In this regard, effective and safe antimicrobials to control food contamination by C. perfringens are needed. Currently, there are very few interventions targeted toward the inactivation of bacteria on food. Most of the available interventions involve heating or organic acids, which can adversely modify the taste and quality of the products. Recently, Schulz et al. (2015) described potential application of plant-produced colicins to control enterohaemoragic E. coli in food. EP 2 143 729 A1 also discloses an endolysin for the treatment, prevention or diagnosis of Clostridium contamination of foodstuff, of food processing equipment, of food processing plants, or of surfaces coming into contact with foodstuff. Bacteriophages, natural enemies of bacteria, have been used during the early part of the 20th century, mostly as therapeutic agents before the discovery of antibiotics. The growing problem of antibiotic resistant pathogens renewed interest in bacteriophage therapy and applications in other fields, where bacterial contamination is of concern, including food treatment. Phage cocktails (Listex P100, SalmoFresh, ListShield) developed by Intralytix have been commercialized. The same company also developed and licensed out the phage product INT-401 ™ for veterinary application, for controlling of Clostridium perfringens in poultry (Miller et al., 2010). The inventors considered that bacteriophage lysins may be even safer alternatives compared to whole bacteriophages and conceived that bacteriophage lysins used as food additives or used for food processing could be a viable solution for controlling Clostridium perfringens and other Clostridia. Antimicrobial activity of several E. coli-produced C. perfringens lysins such as Ply3626, PlyCP26F, PlyCP39O, psm, PlyCM and CP25L has been described (Zimmer et al. 2002; Simmons et al., 2010; Nariya et al., 2011; Schmitz et al., 2011; Gervasi et al., 2014). Departing from the prior art, it is an object of the invention to provide a method and an agent for preventing or reducing contamination of an object such as food or animal feed with Clostridium, notably with Clostridium perfringens. It is another object to provide a process of producing a composition comprising at least one endolysin active against Clostridium. It is a further object to provide a composition active against Clostridium, and a method of treating an infection with Clostridium such as Clostridium difficile or Clostridium perfringens. SUMMARY OF THE INVENTION The present invention provides the following: (1) A method of preventing or reducing contamination of food with Clostridium, comprising contacting said food with a composition comprising at least one endolysin active against said Clostridium, wherein at least one of said at least one endolysin is ZP173 comprising the amino acid sequence of SEQ ID NO: 2 or a derivative of this endolysin, andwherein said derivative or endolysin has a sequence identity of at least 90% to the amino acid sequence of SEQ ID NO: 2; or said derivative or endolysin has from 1 to 30 amino acid substitutions, insertions and/or deletions compared to the amino acid sequence of SEQ ID NO: 2.(2) The method according to item 1, wherein said composition is or comprises a plant mat