Bacteriophage for the treatment of bacterial biofilms

• US 20060140911A1
• Filed: 01/12/2004
• Published: 06/29/2006
• Est. Priority Date: 01/10/2003
• Status: Active Grant

• Current Assignee: Secretary of State for Health and Social Care
• Original Assignee: Health Protection Agency (Government of United Kingdom)
• Inventors: Hart, Anthony, Hughes, Gavin, Sharp, Richard, Walker, James Taggart
• Granted Patent: US 7,758,856 B2
• Time in Patent Office: Days
• US Class Current: 424/93.600
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/76 Viruses; Subviral particles; Bacteriophages
• CPC Class Codes: A61K 38/51 Lyases (4)
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C12N 2795/00032 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 2795/10032 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 2795/10043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12Y 402/02003 Poly(beta-D-mannuronate) lyase (4.2.2.3)

Abstract

The present invention provides a composition for treating a bacterial biofilm, comprising a first bacteriophage that is capable of infecting a bacterium within said biofilm, and a first polysaccharide lyase enzyme that is capable of degrading a polysaccharide within said biofilm. The composition preferably further comprises a pharmaceutically-acceptable antimicrobial agent, and may also include a DNase. Also provided are modified bacteriophages, methods of creating modified bacteriophages, use of said compositions and bacteriophage for treating biofilms, and methods for treating biofilms using the bacteriophages and compositions of the invention.

Claims

1-40. -40. (canceled)

41. A composition for treating a bacterial biofilm, wherein the biofilm is a lung biofilm of a cystic fibrosis patient, comprising a first bacteriophage that is capable of infecting a bacterium within said biofilm, a first polysaccharide lyase enzyme that is capable of degrading a polysaccharide within said biofilm, and a pharmaceutically-acceptable antimicrobial agent.

42. A composition according to claim 41, wherein the pharmaceutically-acceptable antimicrobial agent is an antibiotic or a defensin.

43. A composition according to claim 41, further comprising a DNase.

44. A composition according to claim 41, further comprising a second polysaccharide lyase, wherein the first and second polysaccharide lyase are different.

45. A composition according to claim 41, wherein the first polysaccharide lyase is encoded by the bacteriophage.

46. A composition according to claim 41, wherein the bacteriophage encodes one or more of a pharmaceutically-acceptable antimicrobial agent, a DNase, or a second polysaccharide lyase that is different from the first polysaccharide lyase.

47. A composition according to claim 41, comprising a second bacteriophage, which is different from the first bacteriophage, and wherein the second bacteriophage optionally encodes a second polysaccharide lyase.

48. A composition according to claim 41, comprising a second pharmaceutically-acceptable antimicrobial agent.

49. A composition according to claim 41, wherein the biofilm comprises an opportunistic bacterium.

50. A composition according to claim 41, wherein the first bacteriophage is a GH phage.

51. A composition according to claim 50, wherein the first bacteriophage is a GH bacteriophage encoding a first polysaccharide lyase.

52. A composition according to claim 50, wherein the GH phage is GH4 (ECACC Accession No. 02121203), GH6 (ECACC Accession No. 02121202), GH13 (ECACC Accession No. 02121201), or GH14 (ECACC Accession No. 02121204).

53. A composition according to claim 41, further comprising a second bacteriophage that is a GH phage, wherein the first bacteriophage and second bacteriophage are different.

54. A composition according to claim 41, wherein the first bacteriophage comprises a heterologous gene encoding a first polysaccharide lyase enzyme.

55. A composition according to claim 41, wherein the first and/or second polysaccharide lyase is an alginate lyase.

56. A composition according to claim 41 in the form of an aerosol formulation, comprising one or more of an excipient, surfactant, and/or propellant.

57. A GH bacteriophage selected from the group consisting of GH4 (ECACC Accession No. 02121203), GH6 (ECACC Accession No. 02121202), GH13 (ECACC Accession No. 02121201), and GH14 (ECACC Accession No. 02121204).

58. A method of treating a biofilm infection, wherein the biofilm is a lung biofilm in a cystic fibrosis patient, comprising administering to the patient:

a first bacteriophage capable of infecting a bacterium within said biofilm;

a first polysaccharide lyase enzyme capable of degrading a polysaccharide within said biofilm; and

a pharmaceutically-acceptable antimicrobial agent.

59. A method according to claim 58, wherein the pharmaceutically-acceptable antimicrobial agent is an antibiotic or a defensin.

60. A method according to claim 58, further comprising a DNase.

61. A method according to claim 58, further comprising a second polysaccharide lyase, wherein the first and second polysaccharide lyase are different.

62. A method according to claim 58, wherein the first polysaccharide lyase is encoded by the bacteriophage.

63. A method according to claim 58, wherein the bacteriophage encodes one or more of a pharmaceutically-acceptable antimicrobial agent, a DNase, or a second polysaccharide lyase that is different from the first polysaccharide lyase.

64. A method according to claim 58, comprising a second bacteriophage, which is different from the first bacteriophage, and wherein the second bacteriophage optionally encodes a second polysaccharide lyase.

65. A method according to claim 58, comprising a second pharmaceutically-acceptable antimicrobial agent.

66. A method according to claim 58, wherein the biofilm comprises an opportunistic bacterium.

67. A method according to claim 58, wherein the first bacteriophage is a GH phage.

68. A method according to claim 58, wherein the first bacteriophage is a GH bacteriophage encoding a first polysaccharide lyase.

69. A method according to claim 58, wherein the GH phage is GH4 (ECACC Accession No. 02121203), GH6 (ECACC Accession No. 02121202), GH13 (ECACC Accession No. 02121201), or GH14 (ECACC Accession No. 02121204).

70. A method according to claim 58, further comprising a second bacteriophage that is a GH phage, wherein the first bacteriophage and second bacteriophage are different.

71. A method according to claim 58, wherein the first bacteriophage comprises a heterologous gene encoding a first polysaccharide lyase enzyme.

72. A method according to claim 58, wherein the first and/or second polysaccharide lyase is an alginate lyase.

73. A method according to claim 58 in the form of an aerosol formulation, comprising one or more of an excipient, surfactant, and/or propellant.

74. A method according to claim 58, wherein following administration the bacterial cell count of the biofilm is reduced by at least one log.

75. A method according to claim 58, wherein the composition or bacteriophage is administered in more than one separate dose.

76. A method according to claim 58, wherein the composition or first bacteriophage is administered in at least three separate doses.

77. A method according to claim 58, wherein the first bacteriophage is administered prior to, simultaneously with, or subsequent to the first polysaccharide lyase.

78. A method according to claim 58, wherein the first bacteriophage is administered prior to, simultaneously with, or subsequent to said pharmaceutically-acceptable antimicrobial agent.

79. A method according to claim 58, wherein the first bacteriophage is administered prior to, simultaneously with, or subsequent to a second polysaccharide lyase that is different from the first polysaccharide lyase.

80. A method according to claim 58, wherein the first bacteriophage is administered prior to, simultaneously with, or subsequent to a second bacteriophage that is capable of infecting a bacterium within the biofilm, wherein said second bacteriophage is different from the first bacteriophage.

81. A method according to claim 58, wherein administration is to the site of infection.

82. A method of making a modified bacteriophage capable of degrading a biofilm, wherein the biofilm is a lung biofilm of a cystic fibrosis patient, comprising:

a) selecting at least one gene encoding a polysaccharide lyase enzyme that degrades a polysaccharide within said biofilm;

b) selecting a bacteriophage that is capable of infecting a bacterial species or strain residing within the biofilm; and

c) introducing at least one of the genes selected in step a) into the bacteriophage nucleic acid.

83. A method according to claim 82, wherein the bacteriophage is selected from the group consisting of GH4 (ECACC Accession No. 02121203), GH6 (ECACC Accession No. 02121202), GH13 (ECACC Accession No. 02121201), GH14 (ECACC Accession No. 02121204);

and a bacteriophage having accession No. ATCC 12055-B1, ATCC 12055-B2, ATCC 12055-B3, ATCC 14205-B1, ATCC 14206-B1, ATCC 14207-B1, ATCC 14209-B1, ATCC 14210-B1, ATCC 14211-B1, ATCC 14212-B1, ATCC 14213-B1, ATCC 14214-B1, ATCC 15692-B2, ATCC 15692-B3, ATCC 25102-B1, ATCC BAA-26-B1, ATCC BM-27-B1, ATCC BAA-28-B1, ATCC BM-28-B2, ATCC BAA-29-B1, ATCC BAA-30-B1, ATCC BM-31-B1, ATCC BMA-47-B1, ATCC BAA-79-B1, ATCC BAA-81-B1, and ATCC BM-81-B2.

84. A method according to claim 82, wherein the method further comprises the step of testing the efficacy of the modified bacteriophage against the biofilm in vitro.

85. A method according to claim 82, wherein the bacteriophage specifically infects an opportunistic bacterium.

86. A method according to claim 82, wherein said at least one gene encodes an alginate lyase.

87. A method of identifying a bacteriophage for use in treatment of a biofilm, wherein the biofilm is a lung biofilm of a cystic fibrosis patient, comprising:

a) identifying a bacteriophage that is capable of infecting a bacterial species or strain within said biofilm; and

b) confirming that said bacteriophage encodes a polysaccharide lyase that degrades a polysaccharide within the biofilm.