GENETICALLY ENGINEERED BIOLOGICAL INDICATOR

• US 20080070231A1
• Filed: 09/20/2006
• Published: 03/20/2008
• Est. Priority Date: 09/20/2006
• Status: Active Grant

• Current Assignee: American Sterilizer Company (Steris Corporation)
• Original Assignee: American Sterilizer Company (Steris Corporation)
• Inventors: Franciskovich, Phillip P., Cregger, Tricia A., Yirava, William A.
• Granted Patent: US 8,895,239 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The disclosed technology relates to a genetically engineered biological indicator, comprising: at least one test organism and at least one reporter gene suitable for producing an indicator enzyme, the reporter gene being taken up by the test organism; and at least one repressor gene that inhibits expression of the reporter gene until the reporter gene is exposed to at least one inducer. A process and an apparatus for using the biological indicator are disclosed.

Claims

1. A genetically engineered biological indicator, comprising:

at least one test organism and at least one reporter gene suitable for producing an indicator enzyme, the reporter gene being taken up by the test organism; and

at least one repressor gene that inhibits expression of the reporter gene until the reporter gene is exposed to at least one inducer.

2. The biological indicator of claim 1 wherein the reporter gene and the repressor gene are taken up by the test organism using at least one plasmid and/or at least one virus.

3. The biological indicator of claim 1 wherein the test organism comprises one or more bacteria, pathogens, gram negative organisms, gram positive organisms, vegetative organisms, viruses, non self-replicating agents, sub-cellular components or products of cells, and/or prions.

4. The biological indicator of claim 1 wherein the test organism comprises bacterial spores.

5. The biological indicator of claim 1 wherein the test organism comprises bacteria of the Bacillus, Geobacillus or Clostridia genera.

6. The biological indicator of claim 1 wherein the test organism comprises Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus subtilis, Bacillus sphaericus, Bacillus anthracis, Bacillus pumilus, Bacillus coagulans, Clostridium sporogenes, Clostridium difficile, Clostridium botulinum, Bacillus subtilis globigii, Bacillus cereus, Bacillus circulans, Escherichia coli, or a mixture of two or more thereof.

7. The biological indicator of claim 1 wherein the test organism comprises fungi, mycobacteria, protozoa, vegetative bacteria, or a mixture of two or more thereof.

8. The biological indicator of claim 1 wherein the test organism comprises Aspergillus niger, Candida albicans, Trichophyton mentagrophytes, Wangiella dermatitis, Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium smegmantis, Mycobacterium terrae, Mycobacterium bovis, Mycobacterium tuberculosis, Giardia lamblia, Cryptosporidium parvum, Aeromonas hydrophila, Enterococcus faecalis, Streptococcus faecalis, Enterococcus faecium, Streptococcus pyrogenes, Escherichia coli, Klebsiella (pneumoniae), Legionella pneumophila, Methylobacterium, Pseudomonas aeruginosa, Salmonella choleraesuis, Helicobacter pylori, Micrococcus radiodurans, Deinococcus radiodurans, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, or a mixture of two or more thereof.

9. The biological indicator of claim 1 wherein the test organism comprises vegetative bacteria, vegetative cells and/or their constituent parts.

10. The biological indicator of claim 1 wherein the test organism comprises Aeromonas hydrophila, Enterococcus faecalis, Streptococcus faecalis, Enterococcus faecium, Streptococcus pyrogenes, Escherichia coli, Klebsiella (pneumoniae), Legionella pneumophila, Methylobacterium, Pseudomonas aeruginosa, Salmonella choleraesuis, Helicobacter pylori, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, or a mixture of two or more thereof.

11. The biological indicator of claim 1 wherein the test organism comprises vancomycin resistant enterococci, methicillin resistant Staphyloccus aureus, Mycobacterium cheloni, or a mixture of two or more thereof.

12. The biological indicator of claim 1 wherein the test organism comprises one or more prions.

13. The biological indicator of claim 1 wherein the reporter gene comprises lacZ, bgaB, xylE, cat, gfp, or a mixture of two or more thereof.

14. The biological indicator of claim 1 wherein an indicator enzyme is produced by the reporter gene, the indicator enzyme comprising beta-D-galactosidase, beta-D-glucosidase, alpha-D-glucosidase, alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase lipase, chloroamphenicol acetytransferase, catechol-2,3-dioxygenase, myristate lipase, leucine aminopeptidase, valine aminopeptidase, chymotrypsin, phosphohydrolase, alpha-D-galactosidase, alpha-L-arabinofuranosidase, N-acetyl-beta-glucosaminidase, beta-D-cellobiosidase, alanine aminopeptidase, proline aminopeptidase, tyrosine aminopeptidase, phenylalanine aminopeptidase, beta-D-glucuronidase, fatty acid esterase, or a mixture of two or more thereof.

15. The biological indicator of claim 1 wherein the reporter gene comprises lacZ and the indicator enzyme comprises beta-D-galactosidase.

16. The biological indicator of claim 1 wherein the repressor gene comprises xylR, lacI, tetR, or a mixture of two or more thereof.

17. The biological indicator of claim 2 wherein the plasmid comprises a reporter gene and a repressor gene.

18. The biological indicator of claim 2 wherein the plasmid comprises a lacZ gene and an xylR gene.

19. The biological indicator of claim 2 wherein the plasmid comprises a circular double-stranded DNA.

20. The biological indicator of claim 2 wherein the plasmid is linear.

21. The biological indicator of claim 2 wherein the size of the plasmid is in the range from about 2000 to about 20000 base pairs.

22. The biological indicator of claim 2 wherein from 1 to about 3000 copies of the same plasmid are taken up by a single cell of the test organism.

23. The biological indicator of claim 2 wherein the plasmid comprises one or more DNA sequences that serves as an origin of replication.

24. The biological indicator of claim 2 wherein the plasmid comprises one or more genetic markers.

25. The biological indicator of claim 2 wherein the plasmid comprises one or more multiple cloning sites.

26. The biological indicator of claim 2 wherein the plasmid comprises one or more genes that provide a selective marker to induce the test organism to retain the plasmid.

27. The biological indicator of claim 2 wherein the plasmid comprises at least one origin of replication, at least one selectable marker gene, at least one inducible promoter and at least one reporter gene.

28. The biological indicator of claim 2 wherein the plasmid comprises two origins of replication.

29. The biological indicator of claim 2 wherein the plasmid comprises a gram negative origin of replication and a gram positive origin of replication.

30. The biological indicator of claim 20 wherein the gram negative origin of replication comprises Escherichia coli and the gram positive origin or replication comprises Bacillus subtilis, Geobacillus stearothermophilus, Bacillus atrophaeus, or a mixture of two or more thereof.

31. The biological indicator of claim 2 wherein the plasmid comprises an antibiotic resistance gene and/or a gene with exogenous nutritional capability.

32. The biological indicator of claim 2 wherein the plasmid comprises a chloramphenicol, ampicillin or spectinomycin antibiotic gene, and/or a xylose, lactose or amino acid nutritional gene.

33. The biological indicator of claim 2 wherein the virus comprises at least one gene transporter that comprises nucleic acid surrounded by a capsid.

34. The biological indicator of claim 2 wherein the virus comprises at least one bacteriophage.

35. The biological indicator of claim 2 wherein the virus comprises lambda or M13 bacteriophage.

36. The biological indicator of claim 1 wherein the inducer comprises xylose, allolactose, isopropyl thiogalactoside, metalothionine, or a mixture of two or more thereof.

37. A sterilization process, comprising:

exposing an article to be sterilized and the biological indicator of claim 1 to a sterilization medium.

38. The process of claim 37 wherein the sterilization medium comprises steam, dry heat, radiation, plasma, one or more gaseous sterilants and/or one or more liquid sterilants.

39. The process of claim 37 wherein the sterilization medium comprises electron beam radiation, electromagnetic radiation, gamma radiation, beta radiation, ethylene oxide, gaseous hydrogen peroxide, liquid hydrogen peroxide, formalin, glutaraldehyde, and/or peracetic acid.

40. A process for determining the effectiveness of sterilization, comprising:

exposing at least one article to be sterilized and a sterilization indicator comprising the biological indicator of claim 1 to a sterilization medium; and

contacting the sterilization indicator with at least one inducer and at least one enzyme substrate to determine the effectiveness of the sterilization.

41. The process of claim 40 wherein the inducer comprises xylose, allolactose, isopropyl thiogalactoside, metalothionine, or a mixture of two or more thereof.

42. The process of claim 40 wherein the enzyme substrate comprises:

one or more fluorogenic 4-methylumbelliferyl derivatives;

one or more derivatives of 7-amido-4-methyl-coumarin;

one or more diacetylfluorescein derivatives;

fluorescamine;

or a mixture of two or more thereof.

43. The process of claim 40 wherein the enzyme substrate comprises:

4-methylumbelliferyl-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-lucopyranoside;

4-methylumbelliferyl acetate;

4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide;

4-methylumbelliferyl-N-acetyl-alpha-D-glucosaminide;

4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide;

2′

-(4-methylumbelliferyl)-alpha-D-N-acetyl neuraminic acid;

4-methylumbelliferyl-alpha-L-arabinofuranoside;

4-methylumbelliferyl alpha-L-arabinoside;

4-methylumbelliferyl butyrate;

4-methylumbelliferyl-beta-D-cellobioside;

methylumbelliferyl-beta-D-N,N′

-diacetyl chitobioside;

4-methylumbelliferyl elaidate;

4-methylumbelliferyl-beta-D-fucoside;

4-methylumbelliferyl-alpha-L-fucoside;

4-methylumbelliferyl-beta-L-fucoside;

4-methylumbelliferyl-alpha-D-galactoside;

4-methylumbelliferyl-beta-D-galactoside;

4-methylumbelliferyl-alpha-D-glucoside;

4-methylumbelliferyl-beta-D-glucoside;

4-methylumbelliferyl-beta-D-glucuronide;

4-methylumbelliferyl p-guanidinobenzoate;

4-methylumbelliferyl heptanoate;

4-methylumbelliferyl-alpha-D-mannopyranoside;

4-methylumbelliferyl-beta-D-mannopyranoside;

4-methylumbelliferyl oleate;

4-methylumbelliferyl palmitate;

4-methylumbelliferyl phosphate;

4-methylumbelliferyl propionate;

4-methylumbelliferyl stearate;

4-methylumbelliferyl sulfate;

4-methylumbelliferyl-beta-D-N,N′

, N″

-triacetylchitotriose;

4′

-methylumbelliferyl 2,3,5-tri-beta-benzoyl-alpha-L-arabinofuranoside;

4-methylumbelliferyl-beta-trimethylammonium cinnamate chloride;

4-methylumbelliferyl-beta-D-xyloside;

or a mixture of two or more thereof.

44. The process of claim 40 wherein the enzyme substrate comprises:

L-alanine-7-amido-4-methylcoumarin;

L-proline-7-amido-4-methylcoumarin;

L-tyrosine-7-amido-4-methylcoumarin;

L-leucine-7-amido-4-methylcoumarin;

L-phenylalanine-7-amido-4-methylcoumarin;

7-glutaryl-phenylalanine-7-amido-4-methylcoumarin;

or a mixture of two or more thereof.

45. The process of claim 40 wherein the enzyme substrate comprises:

N-t-BOC-lle-Glu-Gly-Arg 7-amido-4-methylcoumarin;

N-t-BOC-Leu-Ser-Thr-Arg 7-amido-4-methylcoumarin;

N-CBZ-Phe-Arg 7-amido-4-methylcoumarin;

Pro-Phe-Arg 7-amido-4-methylcoumarin;

N-t-BOC-Val-Pro-Arg 7-amido-4-methylcoumarin;

N-glutaryl-Gly-Arg 7-amido-4-methylcoumarin;

or a mixture of two or more thereof.

46. The process of claim 40 wherein the enzyme substrate comprises fluorescein diacetate, fluorescein di-(beta-D-galacto-pyranoside), fluorescein dilaurate, or a mixture of two or more thereof.

47. The process of claim 40 wherein:

the enzyme comprises alpha-D-glucosidase, chymotrypsin, or fatty acid esterase; and

the enzyme substrate comprises 4-methylumbelliferyl-alpha-D-glucoside, 7-glutarylphenylalanine-7-amido-4-methyl coumarin, or 4-methylumbelliferyl heptanoate.

48. The process of claim 40 wherein:

the enzyme comprises alpha-L-arabinofuranosidase; and

the enzyme substrate comprises 4-methylumbelliferyl-alpha-L-arabinofuranoside.

49. The process of claim 40 wherein:

the enzyme comprises beta-D-glucosidase; and

the enzyme substrate comprises 4-methylumbelliferyl-beta-D-glucoside.

50. The process of claim 40 wherein:

the enzyme substrate comprises one or more 5-bromo-4-chloro-3-indolyl derivatives;

one or more nitrophenyl derivatives;

one or more indoxyl derivatives;

one or more and phenolphthalein derivatives;

or a mixture of two or more thereof.

51. The process of claim 40 wherein the enzyme substrate comprises 5-bromo-6-chloro-3-indolyl acetate, 5-bromo-4-chloro-3-indolyl acetate, 5-bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-1,3-diacetate, 5-bromo-4-chloro-3-indolyl-beta-D-fucopyranoside, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, 5-bromo-4-chloro-3-indolyl phosphate, 5-bromo-4-chloro-3-indolyl sulfate, or a mixture of two or more thereof.

52. The process of claim 40 wherein the enzyme substrate comprises:

one or more p-nitrophenol derivatives;

one or more o-nitrophenol derivatives;

or a mixture of two or more thereof.

53. The process of claim 40 wherein the enzyme substrate comprises:

diethyl-p-nitrophenyl phosphate;

di-p-nitrophenyl phosphate;

p-nitrophenyl-2-acetamido-2-deoxy-3-O-beta-galactopyranosyl-beta-gluco pyranoside;

p-nitrophenyl-2-acetamido-2-deoxy-beta-glucopyranoside;

p-nitrophenyl acetate;

p-nitrophenyl-N-acetyl-beta-D-glucosaminide;

p-nitrophenyl-beta-D-N,N′

-diacetylchitobioside;

p-nitrophenyl-alpha-glucopyranoside;

p-nitrophenyl-alpha-maltoside;

p-nitrophenyl-beta-maltoside;

p-nitrophenyl-alpha-mannopyranoside;

p-nitrophenyl-beta-mannopyranoside;

p-nitrophenyl myristate;

p-nitrophenyl palmitate;

p-nitrophenyl phosphate;

bis(p-nitrophenyl)phosphate;

tris(p-nitrophenyl)phosphate;

p-nitrophenyl-beta-glucopyranoside;

p-nitrophenyl-beta-glucuronide;

alpha-p-nitrophenylglycerine;

p-nitrophenyl-alpha-rhamnopyranoside;

p-nitrophenyl stearate;

p-nitrophenyl sulfate;

p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-glucosaminide;

p-nitrophenyl thymidine mono-phosphate;

p-nitrophenyl-2,3,4-tri-O-acetyl-beta-glucuronic acid methyl ester;

p-nitrophenyl valerate;

or a mixture of two or more thereof.

54. The process of claim 40 wherein the enzyme substrate comprises:

o-nitrophenyl acetate;

o-nitrophenyl-beta-glucoside;

o-nitrophenyl-beta-D-glucopyranoside;

nitrophenyl-alpha-galactopyranoside;

nitrophenyl-beta-galactopyranoside;

nitrophenyl butyrate;

nitrophenyl caprate;

nitrophenyl caproate;

nitrophenyl caprylate;

nitrophenyl laurate;

nitrophenyl propionate;

indoxyl-acetate;

indoxyl beta-D-glucoside;

3-indoxyl sulfate;

3-indoxyl phosphate;

phenolphthalein dibutyrate;

phenolphthalein diphosphate;

phenolphthalein disulfate;

phenolphthalein glucuronic acid;

phenolphthalein mono-beta-glucosiduronic acid;

phenolphthalein mono-beta-glucuronic acid; and

phenolphthalein mono-phosphate;

1-diazo-4-benzoylamino-2;

5-diethoxybenzene;

1-diazo-4-benzoylamino-2;

5-diethoxybenzene;

p-diazo-2,5-diethoxy-N-benzoyalanine;

4-chloro-2-methylbenzene diazonium chloride;

o-aminoazotoluene diazonium salt;

1-naphthyl-N-acetyl-beta-D-glucosaminide;

2-naphthyl-phosphate;

2-naphthyl-butyrate;

2-naphthyl-caprylate;

2-naphthyl-myristate;

L-leucyl-2-naphthylamide;

L-valyl-2-naphthylamide;

L-cystyl-2-naphthylamide;

N-benzoyl-DL-arginine-2-naphthylamide;

N-glutaryl-phenylalanine 2-naphthyl-amine;

2-naphthyl-phosphate;

6-Br-2-naphthyl-alpha-D-galacto-pyranoside;

2-naphthyl-beta-D-galacto-pyranoside;

2-naphthyl-2-D-glucopyranoside;

6-bromo-2-naphthol-beta-D-glucopyranoside;

6-bromo-2-naphthyl-2-D-mannopyranoside;

2-naphthyl-alpha-L-fucopyranoside;

naphthyl-AS-BI-phosphate;

naphthyl-AS-BI-beta-D-glucuronide;

or a mixture of two or more thereof.

55. The process of claim 40 wherein the enzyme comprises β

-galactosidase, and the enzyme substrate comprises 5-bromo-4-chloro-3-indolyl-β

-D-galactopyranoside.

56. The process of claim 40 wherein the enzyme comprises β

-galactosidase, and the enzyme substrate comprises 4-methylumbelliferone-β

-D-galactopyranoside.

57. The process of claim 40 wherein the indicator enzyme comprises β

-galactosidase, and the enzyme substrate comprises 5-Bromo-4-chloro-3-indolyl-β

-D-galactopyranoside, 5-Bromo-6-chloro-3-indolyl-β

-galactopyranoside), 5-Bromo-3-indolyl-β

-D-galactopyranoside, 6-Bromo-2-naphthyl-β

-D-galactopyranoside, 6-chloro-3-indolyl-β

-D-galacotpyranoside, 3-Indoxyl-β

-D-galactopyranoside, 5-Iodo-3-indoxyl-β

-D-galactopyranoside, N-methylindoxyl-β

-D-galactopyranoside, 2-Nitrophenyl-β

-D-galactopyranoside, 4-Nitrophenyl-β

-D-galactopyranoside, Phenyl-β

-D-galactopranoside, 2-Chloro-4-nitrophenyl-β

-D-lactoside, 4-methylumbelliferyl-β

-D-galactopyranoside, 4-trifluoromethylumbelliferyl-β

-D-galactopyranoside, Fluorescein di(β

-D-galactopyranoside), Fluorescein mono-β

-D-galactopyranoside, Fluorescein di-(β

-D-acetyl galactosamine), 4-Methylumbelliferyl-β

-D-lactopyranoside, 2-Napthyl-β

-D-galactopyranoside, 8-Hydroxyquinoline-β

-D-galactopyranoside, Resorufin β

-D-galactopyranoside, 3-Carboxyumbelliferyl-β

-D-galactopyranoside, 4-Chloromethyl-6,8-difluoroumbelliferyl-β

-D-galactopyranoside, 6,8-Difluor-4-methylumbelliferyl-β

-D-galactopyranoside, 6,8-Difluoro-4-heptadecylumbelliferyl-β

-D-galactopyranoside, 5-(Pentafluorobenzoylamino)-fluorescein-β

-D-galactopyranoside, C₂-fluorescein-β

-D-galactopyranoside, C₈-fluorescein-β

-D-galactopyranoside, C₁₂-fluorescein-β

-D-galactopyranoside, 5-Chloromethylfluorescein-β

-D-galactopyranoside, C₁₂-resorufin-β

-D-galactopyranoside, and/or 7-Hydroxyl-9H-(1,3-dichlor-9,9-dimethylacridin-2-one).

58. A sterilization indicator, comprising:

a carrier, the carrier having a first surface and a second surface;

a support, the support having a first section and a second section, the carrier overlying the first section of the support, the second surface of the carrier being adhered to the first section of the support; and

the biological indicator of claim 1 being supported by the carrier, the second section of the support being of sufficient dimension to permit handling the sterilization indicator without contacting the biological indicator.

59. A sterilization indicator, comprising:

a first compartment containing the biological indicator of claim 1, the first compartment being adapted to permit the biological indicator to be brought into contact with a sterilization medium during sterilization; and

a second compartment containing at least one inducer and at least one enzyme substrate, the second compartment being adapted to maintain the inducer and enzyme substrate separate from the biological indicator during sterilization, and the second compartment being adapted to permit the inducer and enzyme substrate to contact the biological indicator after the biological indicator has been exposed to the sterilization medium.