WO2020136613A1 - Indicateur biologique de lecture instantanée avec confirmation de croissance - Google Patents

Indicateur biologique de lecture instantanée avec confirmation de croissance Download PDF

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Publication number
WO2020136613A1
WO2020136613A1 PCT/IB2019/061398 IB2019061398W WO2020136613A1 WO 2020136613 A1 WO2020136613 A1 WO 2020136613A1 IB 2019061398 W IB2019061398 W IB 2019061398W WO 2020136613 A1 WO2020136613 A1 WO 2020136613A1
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Prior art keywords
spores
sterilization process
indicator
liquid medium
compartment
Prior art date
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PCT/IB2019/061398
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English (en)
Inventor
Joshua D. Erickson
Francois AHIMOU
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3M Innovative Properties Company
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Publication of WO2020136613A1 publication Critical patent/WO2020136613A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/22—Testing for sterility conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/10—DNA staining

Definitions

  • a standard type of biological sterility indicator includes a known quantity of test microorganisms, for example Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) or Bacillus atrophaeus (formerly Bacillus subtilis) spores, which are many times more resistant to a sterilization process than most contaminating organisms. After the indicator is exposed to the sterilization process, the spores are incubated in nutrient medium to determine whether any of the spores survived the sterilization process, with spore growth indicating that the sterilization process was insufficient to destroy all of the microorganisms.
  • Geobacillus stearothermophilus formerly Bacillus stearothermophilus
  • Bacillus atrophaeus originally Bacillus subtilis
  • the activity of an enzyme which can be correlated with spore viability, is determined.
  • a self-contained sterilization process biological indicator is now provided, therein with everything needed to rapidly assess the effectiveness of a sterilization process and subsequently to detect germination and/or outgrowth of viable spores, if present, after exposing the self-contained sterilization process biological indicator to a sterilant gas.
  • this discovery provides its user with the ability to confirm the rapid assessment (by measuring fluorescence of a nucleic acid interacting dye) of inactivation of the spores by the sterilant gas.
  • the aforementioned self- contained sterilization process biological indicator can be used in a rapid method of determine effectiveness of a sterilization process.
  • the rapid method does not require any germination or growth of the test microorganism, the effectiveness of the sterilization process can be determined almost immediately after the sterilization process has been completed.
  • the optional confirmation can be conducted if the rapid result indicates all of the spores are inactivated or if the rapid result indicates not all of the spores are inactivated.
  • the present disclosure provides a self-contained sterilization process biological indicator.
  • the indicator can comprise a cuvette having at least one liquid-impermeable wall that forms an opening into a compartment, a nucleic acid-interacting dye disposed in the compartment, a predetermined number of sterilization process-resistant spores disposed in the compartment, a first liquid medium disposed in the compartment, and a nutrient composition that facilitates germination and/or outgrowth of a viable spore.
  • the spores in the self-contained sterilization process biological indicator are contacting the dye.
  • the nutrient composition is disposed in the compartment and isolated from the spores.
  • the opening is part of a pathway that permits passage of a sterilant gas from outside the cuvette into the compartment.
  • the nucleic acid-interacting dye is fluorescent when it interacts with DNA or RNA.
  • the self-contained sterilization process biological indicator further can comprise a container disposed in the
  • a detection reagent, an enhancer reagent, or a collisional quenching reagent can be disposed in the compartment.
  • the liquid medium can be disposed in the container.
  • the nucleic acid-interacting dye can be selected from the group consisting of Acridine Orange, SYTO 9, SYTO 16, DAPI, propidium iodide, and a combination of any two or more of the foregoing nucleic acid-interacting dyes.
  • the spores can be produced by a species of microorganisms selected from the group consisting of Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus megaterium, Clostridium sporogenes, Bacillus coagulans, and a combination of any two or more of the foregoing species.
  • the present disclosure provides a method of determining effectiveness of a sterilization process.
  • the method can comprise positioning the self-contained sterilization process biological indicator of any one of the above embodiments in a sterilization chamber; while the indicator is positioned in the sterilization chamber, exposing the indicator to a sterilant gas; contacting the spores with the liquid medium; contacting the spores with the nutrient composition; after contacting the spores with the nutrient composition, incubating the indicator at a predetermined temperature for a period of time sufficient to permit germination and at least one cell division of a germinated spore; after the exposing the sterilization process biological indicator to the sterilant gas and after the contacting the spores with the liquid medium and before the incubating the indicator at the predetermined temperature, measuring a first fluorescence intensity emitted by the nucleic acid- interacting dye in the sterilization process biological indicator; comparing the first fluorescence intensity to a reference fluorescence intensity to determine whether the
  • the liquid medium can be contained in an container that is disposed in the container.
  • the container is impermeable to the aqueous liquid medium.
  • the method further can comprise disintegrating the container to contact the spores with the liquid medium.
  • the method further can comprise prior to the exposing the indicator to the sterilant gas, positioning an article to be sterilized in the sterilization chamber.
  • biological sterilization process indicators “sterilization process biological indicator”,“sterilization process indicator”,“biological indicator”,“BI”,“indicator”,“self- contained biological indicator”, and“SCBI” are used interchangeably.
  • the phrases “substantially dry”, “substantially water- free” or the like refer to a composition or a coating which has a water content no greater than about the water content of the dehydrated coating once it has been permitted to equilibrate with the ambient environment.
  • E5, E6, and E7 are used interchangeably herein with 10 5 , 10 6 , and 10 7 , respectively.
  • FIG. 1 is a partially-exploded, cross-sectional schematic view of one embodiment of a self- contained sterilization process biological indicator according to the present disclosure.
  • FIG. 2 is a top view of the self-contained sterilization process biological indicator of FIG. 1.
  • FIG. 3 is a partially-exploded, cross-sectional schematic view of an alternative embodiment of a self-contained sterilization process biological indicator according to the present disclosure.
  • self-contained biological sterilization process indicators are now provided which can detect an indication of viable spores almost immediately upon removal of the biological sterilization process indicators from a sterilization process.
  • the near- immediate detection of an indication of viable spores (after removal of the BI from a sterilizer) eliminates undesirably-long waiting periods to determine whether a sterilization process was effective.
  • the indication of viable spores present in the biological sterilization process indicators can be detected automatically, thereby eliminating a potential human error in deciding whether microbial growth or enzyme activity is present after the biological sterilization process indicators have been exposed to a sterilization process.
  • the rapid result (either indicating sterility or non-sterility) subsequently can be confirmed (e.g., by detecting germination and growth of a viable spore) using a self-contained biological sterilization process indicator of the present disclosure according to a method of the present disclosure.
  • the at least one nucleic acid-interacting dye can interact with nucleic acids present in the spores, resulting in an increase in fluorescence intensity in the biological indicator.
  • the interaction of the dye with the nucleic acids while the spores are exposed to a sterilant or shortly thereafter provides an early indication of viable spores, if present in the biological indicator after the exposure.
  • the at least one nucleic acid-interacting dye can interact with nucleic acids present in the plurality of spores to produce a measurable fluorescence intensity that corresponds to killed spores.
  • the investigators believe this is due to the ability of the dye to penetrate spores that have increased permeability (e.g., due to poor integrity of the spore coat, the spore cortex, and/or the spore membrane(s) (e.g., spore inner membrane and/or spore outer membrane) after exposure to a sterilant gas) of the nonviable spores. This effect is not seen in vegetative cells.
  • the SYT09 nucleic acid-interacting dye can penetrate both live and dead vegetative cells, as evidenced by the LIVE/DEAD® 5acLightTM Bacterial Viability Kit available from Molecular Probes, Inc. (Eugene, OR). Selective interaction of the dye with nucleic acids present in the nonviable spores results in lower fluorescence when viable spores are present. Thus, an absence of viable spores in the biological indicator after exposure to a sterilant gas results in a correspondingly higher fluorescence intensity.
  • a number of sterilization process are presently known and in use, including, for example, exposure to steam, dry heat, gaseous or liquid agents such as ethylene oxide, hydrogen peroxide, peracetic acid, and ozone, and radiation.
  • the plurality of sterilization process resistant spores may be selected according to the sterilization process to be used. Any spores may be used as long as they provide sufficient resistance to the sterilization process conditions, such that the spores are more resistant to the sterilization process conditions than most microorganisms encountered in natural contamination. For example, for a steam sterilization process, Gb.
  • the plurality of sterilization process resistant spores is selected from the group consisting of Geobacillus stearothermophilus, Bacillus atrophaeus, Bacillus megaterium, Clostridium sporogenes, Bacillus coagulans, and a combination thereof.
  • the plurality of sterilization process resistant spores is selected from the group consisting of Gb. stearothermophilus, B. atrophaeus, B. megaterium, and a combination thereof.
  • the present disclosure describes the microorganisms used in the biological sterilization indicator as being “spores;” however, it should be understood that the type of microorganism (e.g., spore) used in a particular embodiment of the biological sterilization indicator is selected for being highly resistant to the particular sterilization process contemplated. Accordingly, different embodiments of the present disclosure may use different microorganisms, depending on the sterilization process for which the particular embodiment is intended.
  • the present biological indicators and methods can detect the presence of nucleic acids within the spores, without the need for germination and/or outgrowth.
  • a sub-lethal concentration of nucleic acid-interacting dye is used so that germination and/or outgrowth of the spores can be done to confirm that the microorganisms in the biological indicators of the present invention have been inactivated (e.g., killed).
  • the concentration of the nucleic acid interacting fluorescent dye in the medium is not more than 0.10 mM, 0.05 mM, or 0.01 mM.
  • the concentration of the dye is not less than 0.0001 mM, 0.0005 mM, or 0.001 mM.
  • an elevated temperature for example, 50° C, 100° C, 121° C, 132° C, 134° C, 134° C, or the like, is included or may be encountered in the process. Accordingly, for certain embodiments, including any one of the above biological indicator and method embodiments, the nucleic acid-interacting fluorescent dyes are stable at sterilization temperatures.
  • nucleic acid-interacting fluorescent dye is stable at a temperature up to at least 121° C.
  • the nucleic acid-interacting fluorescent dye is stable at a temperature up to at least 132° C.
  • the nucleic acid interacting fluorescent dye is stable at a temperature up to at least 134° C.
  • the nucleic acid-interacting fluorescent dye is stable at a temperature up to at least 135° C.
  • the nucleic acid-interacting fluorescent dye is stable when exposed to temperatures between 121-135° C for periods of time that are customary for sterilization processes.
  • the liquid medium is essentially free of any background fluorescence at emission and excitation wavelengths used to detect the fluorescence intensity of the nucleic acid interacting dye when it is interacting with nucleic acid in the biological indicator. This may provide an improved sensitivity because any background level of fluorescence occurring at the same wavelengths as the emission and excitation of the dye interacting with a nucleic acid, is minimized.
  • the medium is essentially free of any nucleic acids other than nucleic acids present in and produced by the plurality of spores. This may provide an improved sensitivity because any baseline level of fluorescence resulting from the dye interacting with any nucleic acids not present in the plurality of spores is minimized.
  • the nucleic acid-interacting dye has a lower level of fluorescence at the emission wavelength (or wavelength range) when not interacting with a nucleic acid and a higher level fluorescence at this wavelength when interacting with a nucleic acid.
  • the nucleic acid-interacting dye when not interacting with a nucleic acid the nucleic acid-interacting dye has a fluorescence quantum yield of less than 0.1, preferably less than 0.05, more preferably not more than 0.01.
  • the nucleic acid interacting dye when interacting with a nucleic acid has a fluorescence quantum yield of at least 0.1, preferably at least 0.2, more preferably at least 0.4.
  • the nucleic acid-interacting fluorescent dye is a dye which interacts with DNA, RNA, or DNA and RNA.
  • the interaction of the dye with DNA and RNA may be the same or different.
  • the dye interacting with DNA may have a different excitation and/or emission maximum than the same dye interacting with RNA.
  • the nucleic acid-interacting fluorescent dye is a dye which interacts with the nucleic acids in a variety of ways know in the art, including intercalation, electrostatic attraction, charge interaction, hydrophilic-hydrophobic interaction, or a combination thereof.
  • nucleic acid-interacting fluorescent dye is selected from the group consisting of acridine orange, a substituted unsymmetrical cyanine dye, and salts thereof, and combinations thereof.
  • Acridine orange bound to a DNA has an excitation maximum at about 490 nm and an emission maximum at about 520 maximum, but when bound to an RNA about 530 nm and 620 nm, respectively. See Maclnnes, J. W and McClintock, M., Differences in Fluorescence Spectra of Acridine Orange-DNA Complexes Related to DNA Base Composition, Biopolymers.
  • fluorescence at the emission maximum of the dye bound to RNA can be used to indicate a presence of viable spores.
  • Suitable examples of substituted unsymmetrical cyanine dyes include dyes available under the trade name, SYTO (Invitrogen Corp., Carlsbad, Calif.). SYTO dyes are permeable to many, if not all, cell membranes but may differ from each other, for example, in degree of permeability through intact spore coats and/or spore cortexes, and/or spore membranes; amount of fluorescence intensity increase when bound to a nucleic acid; excitation and emission maxima; selectivity in binding to DNA and RNA; and binding affinity to DNA and RNA. See Tamok, Cytometry Part A, 73A, 477-479 (2008).
  • Suitable nucleic acid-interacting dyes of the present disclosure include those nucleic acid-interacting dyes that are substantially excluded from viable spores (i.e., spores that are capable of germination to form vegetative cells that are capable of reproduction) but are not excluded from nonviable spores (e.g., spores that have been exposed to a lethal sterilization process.
  • the determination of selective permeability of any given nucleic acid-interacting dye is easily performed by a person having ordinary skill in the art by separately contacting similar numbers of viable and nonviable spores of a given species with the nucleic acid-interacting dye in a microwell plate according to the method described in Example 2 of U.S. Provisional Patent Application No. 62/785,386, filed on even date herewith and entitled“INSTANT READ-OUT BIOLOGICAL INDICATOR”, which is incorporated herein by reference in its entirety.
  • Suitable nucleic acid-interacting dyes include, but are not limited to Acridine Orange, SYT09, SYTO 16, DAPI, propidium iodide, and a combination of any two or more of the foregoing nucleic acid-interacting dyes.
  • the substituted unsymmetrical cyanine dye is SYT09 or SYTO 16.
  • Preferred nucleic acid-interacting dyes penetrate the spore coat and/or spore cortex and/or spore membranes substantially better after the spores have been exposed to a gas sterilant under conditions that result in the disruption and/or activation of substantially all of the spores.
  • the disruption of the spore coat and/or spore cortex and/or spore membrane(s) facilitates penetration of the dye into the spore, thereby also facilitating interaction of the dye with nucleic acids disposed in and/or released from the disrupted spores.
  • the nucleic acid-interacting dyes can be used in a self-contained biological sterilization process indicator at concentrations of about 0.05 mIUI to about 50 mM.
  • the nucleic acid-interacting dye differentially penetrates (and interacts with the nucleic acid in) inactivated (killed) spores relative to viable spores.
  • the higher the number of spores inactivated by the sterilant gas the greater the interaction between the nucleic acid-interacting dye and the nucleic acid in the spores.
  • the higher the number of spores inactivated by the sterilant gas the greater the intensity of fluorescent signal emitted by the dye-bound nucleic acid from the spores in the sterilization process indicator.
  • the at least one nutrient composition of the present disclosure induces germination and may also provide for outgrowth of the spores, if viable, with the simultaneous production of nucleic acids. Moreover, it has been found that the presence of the nutrient composition with the dye reduces the effective toxicity of the dye with respect to the spores.
  • the nutrient composition includes one or more sugars, for example, glucose, fructose, cellibiose, orthe like.
  • the nutrient composition may also include a salt such as potassium chloride, calcium chloride, orthe like.
  • the nutrient composition may also include at least one amino acid, for example, at least one of methionine, phenylalanine, and tryptophan.
  • the germination medium may also include one or more other materials with the nutrient composition.
  • the quantities of such nutrient compositions and materials as well as other nutrient compositions known in the art for inducing germination and initial outgrowth of the sterilization process resistant spores may be used.
  • Media components and concentrations are known and described, for example, WO 99/05310 (Tautvydas) and Zechman et al., J. Food, Sci., 56, 5, p. 1408-1411 (1991), referred to as Zechman and Pflug,
  • the biological indicator further comprises a collisional quenching component disposed in the compartment (e.g., in the liquid medium.
  • a collisional quenching component disposed in the compartment (e.g., in the liquid medium.
  • Such components reduce the background fluorescence signal of free (unbound to nucleic acid) nucleic acid-interacting dye through collisional quenching.
  • species known to collisionally quench fluorescence include organic compounds such as purines, pyrimidines, aliphatic amines, and nitroxides, certain ions, for example, nitrate anions and dissolved metal ions.
  • Other species known to collisionally quench fluorescence are described, for example, in Principles of Fluorescence Spectroscopy, Chapter 9, Joseph R. Lakowicz, Plenum Press, 1983.
  • the biological indicator further comprises at least one reference dye disposed in the compartment (e.g., in the liquid medium).
  • the reference dye does not bind to nucleic acids but responds similarly to nucleic acid-interacting dyes to changes in temperature or changes in the medium induced by spore germination and outgrowth, for example an increase or decrease in pH, ionic strength, or concentration change in metabolic by-products that alter the fluorescent signal.
  • signal from the nucleic acid-interacting dye binding to nucleic acid can be distinguished from signal produced from a change in temperature or a change in the media.
  • the reference dye preferably fluoresces at a different wavelength than the nucleic acid-interacting dye.
  • the sterilization process indicator provided herein comprises a predetermined number of sterilization process resistant viable spores disposed in a compartment of a cuvette.
  • the predetermined number of spores can comprise at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 s , at least 10 6 , or at least 10 7 viable spores.
  • of the present disclosure has a predetermined number of about 10 6 viable spores disposed therein.
  • the germination medium and the sterilization process resistant spores are kept separate but in close proximity to each other for ease of combining the medium and the spores when desired, for example, after exposure to a sterilization process and before detection of a first fluorescence intensity; and then incubating spores with the germination medium to confirm whether or not any viable spores are present as would be indicated by a lower than expected first fluorescence intensity.
  • the plurality of sterilization process resistant spores and the germination medium are separate from each other and adjacent each other in the chamber of the process indicator.
  • the germination medium is an aqueous solution or suspension.
  • concentration of the at least one nucleic acid-interacting fluorescent dye in the medium is dependent on the minimum level needed to provide a detectable first fluorescence intensity after the process indicator is exposed to a sterilant in a sterilization process, and on the maximum level that can be tolerated by the spores without a significant adverse effect on the germination and/or growth of the spores. Examples of dye concentrations that may be used are described above.
  • the detectable first fluorescence intensity can be measured in less than 30 minutes, preferably less than 15 minutes, more preferably in less than 10 minutes, and even more preferably in less than 5 minutes after completion of exposure of the process indicator to the sterilant.
  • the germination medium is in a dry form.
  • the at least one nucleic acid-interacting fluorescent dye and the at least one nutrient composition can be dried together or separately to form a film or a layer on a support film or on a carrier material, optionally in a desired shape, or compounded together or separately as dry solids to form a tablet(s), caplet(s), or capsule(s).
  • any of these forms can be kept adjacent the spores, and water or an aqueous buffer can be added at an appropriate time to incubate the spores with the resulting medium suspension or solution.
  • the resulting medium can be a liquid or a gel. Additional embodiments of a medium in a dry form, which can be used in the indicator and method embodiments described herein are described in PCT Application Publication No . WO2010045138 A2.
  • an incubation temperature above room temperature may be used.
  • the incubation temperature is at least 37° C.
  • the incubation temperature is at least 50° C.
  • the incubation temperature is 50 to 60° C.
  • the sterilization process indicator provided herein comprises a predetermined number of sterilization process resistant viable spores disposed in an compartment of a cuvette.
  • the predetermined number of spores can comprise at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , or at least 10 7 viable spores.
  • of the present disclosure has a predetermined number of about 10 6 viable spores disposed therein.
  • the spores may be disposed on a carrier.
  • the carrier is a sheet material such as paper, woven cloth, nonwoven cloth, plastic, a polymenc material, a microporous polymeric material, metal foil, glass, porcelain, ceramic, or the like, or a combination thereof.
  • the sheet material is water-absorbent or can be wetted to aid in quickly bringing the liquid medium in intimate contact with the spores at the appropriate time.
  • the sterilization process indicator also comprises a container, which contains the liquid medium and, optionally, the nutrient composition, without allowing the sterilant or any microorganism to enter the container.
  • the carrier is adjacent to the container for ease of contacting the spores, supported by the carrier, with the liquid medium and the germination medium when incubation is to be initiated.
  • the container can be readily opened to contact the spores with the medium by expelling a plug, crushing or puncturing the container, or the like.
  • the container can be equipped with a plug, or at least a portion of the container can be a breakable material, such as glass (e.g., glass ampoule) or other material, which can be breached by physical pressure but sufficiently tough to remain intact during
  • FIGS. 1 and 2 show one embodiment of a sterilization process indicator 100 according to the present disclosure.
  • the sterilization process indicator 100 includes a cuvette 10 having at least one liquid impermeable wall (e.g., side wall 12A and bottom wall 12B) that forms an opening 14 into a compartment 15.
  • the cuvette 10 is shown as a circular tube, but other known
  • the at least one wall is preferably transparent or translucent to the extent that electromagnetic radiation of a particular wavelength (e.g., u.v. -visible) and intensity sufficient to cause a nucleic acid-interacting dye to fluoresce can be transmitted therethrough.
  • the walls are preferably transparent or translucent to the extent that electromagnetic radiation at a particular wavelength (e.g., in the visible wavelength range) emitted by the nucleic acid-interacting dye can be transmitted through the wall and be measured.
  • Suitable materials for the walls may include glass, polycarbonate, polypropylene, polyester, and the like.
  • the at least one wall of the cuvette transmits at least 90% of incident
  • electromagnetic radiation within a wavelength range of at least 500 to 700 nm, preferably at least 500 to 675 nm.
  • At least a portion of the cuvette can be configured (e.g., shaped and dimensioned) to be received into an instrument capable of detecting fluorescence emitted by the nucleic acid-interacting dye. That is, the instrument is configured to illuminate the portion of the cuvette with electromagnetic radiation of an appropriate first wavelength to cause fluorescence of the nucleic acid-interacting dye when the dye is bound to DNA or RNA. Moreover, the instrument is configured to detect and quantify electromagnetic radiation of a second wavelength emitted by fluorescence of the nucleic acid-bound nucleic acid-interacting dye.
  • the instrument is also capable of measuring absorbance or scattering of electromagnetic radiation (e.g., at wavelengths typically absorbed or scattered by spores and/or microorganisms) passed through the portion of the cuvette. That is, the instrument is configured to pass the radiation through the portion of the cuvette and also detect an amount of radiation that passed through the cuvette. In certain alternative embodiments, at least a portion of the cuvette is configured (e.g., shaped and dimensioned) to be received into a second instrument, the second instrument being capable of measuring the absorbance of electromagnetic radiation passed through the cuvette.
  • electromagnetic radiation e.g., at wavelengths typically absorbed or scattered by spores and/or microorganisms
  • a liquid medium 16 Disposed in the compartment 15 is a liquid medium 16, a nucleic acid-interacting dye (not shown), and a predetermined number of sterilization process-resistant spores (not shown). Suitable nucleic acid-interacting dyes are disclosed hereinabove.
  • the liquid medium 16 can be a liquid (e.g., an aqueous liquid) in which the nucleic acid-interacting dye is suspended and/or dissolved.
  • the nucleic acid-interacting dye is disposed in the liquid medium.
  • the spores are disposed in the liquid medium.
  • the spores and the nucleic acid-interacting dye are disposed in the liquid medium 16.
  • the sterilization process indicator 100 is exposed to a sterilant gas (e.g., in the sterilization chamber of an automated sterilizer).
  • the sterilant gas passes through the closure member 22 into the compartment 15 where it contacts the liquid medium in which the spores and the nucleic acid-interacting dye are disposed.
  • the nucleic acid-interacting dye is able to penetrate the spore and interact with the nucleic acid therein.
  • the liquid medium, whether present in the process indicator in the compartment or in a container as described herein has a volume of not less than 10 microliters, not less than 20 microliters, not less than 50 microliters, not less than 100 microliters, or not less than 150 microliters. In certain embodiments, the liquid medium, whether present in the process indicator in the compartment or in a container as described herein, has a volume of not more than 1000 microliters, not more than 500 microliters, not more than 400 microliters, not more than 300 microliters, not more than 250 microliters, or not more than 200 microliters. In certain embodiments, the volume of the liquid medium is about 10 microliters to about 1000 microliters. In certain embodiments, the volume of the liquid medium is about 10 microliters to about 500 microliters. In certain embodiments, the volume of the liquid medium is about 100 microliters to about
  • the opening 14 to compartment 15 is provided with a gas-transmissive,
  • microorganism -impermeable closure member 22 which may be adhered to cuvette 10 by an adhesive, a heat seal, or the like. Alternatively, closure member 22 may be held on to opening 14 with a cap 26 having an aperture 28. During exposure to a sterilant gas, the sterilant gas is directed through the closure member 22, enters the compartment 15, and contacts the spores. In certain preferred embodiments, the pathway that permits passage of the sterilant gas into the cuvette also resists or prevents passage into the container of bacteria from outside the sterilization process indicator.
  • suitable materials for closure members include microporous materials such as a filter membrane. Alternative embodiments for the above structures are shown in U.S. Patent Publication No. 20150165082 (Chandrapati et al.), filed Oct. 17, 2008, entitled Biological Sterilization Indicator, System, and Methods of Using Same.
  • a skilled artisan will recognize alternative structures (e.g., a tortuous path) that may be suitable for providing passage of a sterilant gas into the compartment 15 while resisting or preventing passage of bacteria into the compartment.
  • alternative structures e.g., a tortuous path
  • the container can be positioned outside of and the adjacent compartment, with a conduit providing fluid communication between the container and the compartment.
  • Container 18, which is sealed, can be a breakable ampoule, but could alternatively be a container equipped with a plug, or other mechanism which when activated allows the nutrient composition to contact carrier 16 and the spores supported thereon.
  • Container 18 is shown as an elongated ampoule, but other known configurations can be used as well.
  • the dry powder 30 further can comprise the collisional quenching component and/or detection reagent disclosed herein.
  • the aqueous liquid medium whether present in the process indicator in the compartment or in a container as described herein, has a volume of not less than 10 microliters, not less than 20 microliters, not less than 50 microliters, not less than 100 microliters, or mot less than 150 microliters. In certain embodiments, the aqueous liquid medium, whether present in the process indicator in the compartment or in a container as described herein, has a volume of not more than 1000 microliters, not more than 500 microliters, not more than 400 microliters, not more than 300 microliters, not more than 250 microliters, or not more than 200 microliters.
  • the volume of the aqueous liquid medium is about 10 microliters to about 1000 microliters. In certain embodiments, the volume of the aqueous liquid medium is about 10 microliters to about 500 microliters. In certain embodiments, the volume of the aqueous liquid medium is about 100 microliters to about 250 microliters.
  • the opening 14 to compartment 15 is provided with a gas-transmissive,
  • microorganism -impermeable closure member 22 which may be adhered to cuvette 10 by an adhesive, a heat seal, or the like. Alternatively, closure member 22 may be held on to opening 14 with a cap 26 having an aperture 28. During exposure to a sterilant gas, the sterilant gas is directed through the closure member 22, enters the compartment 15, and contacts the spores.
  • suitable materials for closure members include microporous materials such as a filter membrane.
  • Alternative embodiments for the above structures are shown in U.S. Patent Publication No. 20150165082 (Chandrapati et ak), filed Oct. 17, 2008, entitled Biological Sterilization Indicator, System, and Methods of Using Same. A skilled artisan will recognize alternative structures (e.g., a tortuous path) that may be suitable for providing passage of a sterilant gas into the compartment 15 while preventing passage of bacteria into the compartment.
  • the container is opened (e.g., by crushing) prior to exposing the sterilization process indicator to the sterilant gas. In certain embodiments, the container is opened (e.g., by crushing) after exposing the sterilization process indicator to the sterilant gas. Opening the container places the nutrient composition in contact with the aqueous liquid medium, thereby dissolving the nutrient composition into the medium and placing the nutrient composition in fluid contact with the spores.
  • the sterilant gasses include, but are not limited to, water vapor (i.e., steam), ethylene oxide, hydrogen peroxide, and ozone.
  • FIG. 3 shows an alternative embodiment of a sterilization process indicator 200 according to the present disclosure.
  • the indicator comprises a cuvette 10 having at least one liquid impermeable wall (e.g., side wall 12A and bottom wall 12B) that forms an opening 14 into a compartment 15, each as described hereinabove.
  • a liquid medium 16 Disposed in the compartment 15 is a liquid medium 16, a nucleic acid-interacting dye and a predetermined number of sterilization process- resistant spores 20, each as described hereinabove.
  • the liquid medium 16 is contained in a container 18.
  • the sterilization process indicator 200 also includes a gas-transmissive, microorganism-impermeable closure member 22 and a cap 26, each as described hereinabove.
  • the liquid medium 16 in the container 18 can have the nutrient composition (not shown) suspended and/or dissolved therein.
  • the nutrient composition is isolated from the spores.
  • aqueous liquid medium further can have a portion or all of the collisional quenching component and or a portion or all of the detection reagent suspended and/or dissolved therein.
  • Container 18, which holds the liquid medium 16 is shown within compartment 15.
  • Container 18, which is sealed can be a breakable (e.g., plastic or glass) ampoule, but could alternatively be a container equipped with a plug, or other mechanism which when activated (e.g., opened inside the compartment 15) allows the liquid medium 16 to contact the spores 20.
  • Container 18 is shown as a frangible, elongated ampoule, but other known configurations can be used as well.
  • the spores are disposed (e.g., in a substantially water-free layer or coating) on a surface in the compartment.
  • the spores may be suspended in a suitable liquid, which is then deposited onto the surface and subsequently dried (e.g., by evaporation).
  • the surface can be a portion of the inner surface of one of the at least one walls (e.g., the bottom wall) of the cuvette.
  • the predetermined number of process-resistant spores 20 is disposed (e.g., as a layer or a substantially water-free coating) on an optional carrier 24.
  • the spores 20 can be disposed in a composition comprising a plurality of sterilization process-resistant microbial spores and a nucleic acid-interacting dye, wherein the composition is substantially water-free, as described in U.S. Provisional Patent Application No. 62/785,386, filed on even date herewith and entitled“INSTANT READ-OUT BIOLOGICAL INDICATOR”.
  • the enhancer reagent (not shown, described herein) and/or the nucleic acid interacting dye (not shown) can be disposed in the container 15 with the spores (e.g., in the layer or substantially water-free coating).
  • the carrier 24 can be any suitable material onto which the spores 20 can be disposed (e.g., by a coating process) wherein the material does not substantially hinder or prevent 1) contact between the spores and a sterilant gas, 2) contact between the spores and the nucleic acid-interacting dye (e.g., by adsorbing and/or sequestering the dye from contact with the spores), and/or 3) detection of fluorescence by the nucleic acid-interacting dye when it is bound to DNA or RNA (e.g., by substantially absorbing the electromagnetic radiation used to excite the fluorescent dye and/or by substantially absorbing the electromagnetic radiation emitted by the nucleic acid-bound dye).
  • Suitable materials for carriers 24 include, but are not limited to paper, glass, or a polymeric sheet or film.
  • a self-contained sterilization process biological indicator can comprise an enhancer reagent disposed in the compartment.
  • the enhancer reagent contacts the spores (e g ., it may be deposited on the inner surface of the wall or on the carrier with the spores).
  • the enhancer reagent is disposed in the liquid medium and contacts the spores when the liquid medium contacts the spores. The enhancer reagent, when present in the sterilization process indicator, improves the consistency of the sterilization of the spores and/or the detection of viable spores after the sterilization process indicator is exposed to the sterilant gas.
  • the enhancer reagent promotes stability of viable spores during storage and/or facilitates the resuspension of dried spores into the liquid medium and/or facilitates penetration of the nucleic acid-interacting dye into the spores.
  • Nonlimiting examples of suitable enhancer reagents include glycerol, sucrose, trehalose, polyvinyl pyrrolidone, and a combination of any two or more of the foregoing enhancer reagents.
  • a self-contained sterilization process biological indicator of the present disclosure can comprise a buffering agent disposed in the compartment.
  • the buffering agent can be disposed in the liquid medium. Additionally, or alternatively, the buffering agent can be disposed with the spores (e.g., in a dried coating as discussed above).
  • the buffering agent can serve to buffer the liquid medium at a pH that facilitates fluorescence of the nucleic acid interacting dye.
  • Suitable buffering agents include, but are not limited to HEPES buffer (e.g., 50 mM), Tris-EDTA (e.g., 50 mM Tris, 10 mM EDTA) buffer, Tris buffer (e.g., 50 mM).
  • the buffer can be selected for use at a neutral pH (e.g., about 6.0-8.0).
  • a self-contained sterilization process biological indicator of the present disclosure can comprise a collisional quenching component, as described hereinabove, disposed in the compartment.
  • the collisional quenching component may be dissolved and/or suspended in the liquid medium, for example.
  • the sterilization process indicator 200 is exposed to a sterilant gas (e.g., in the sterilization chamber of an automated sterilizer).
  • the sterilant gas passes through the closure member 22 into the compartment 15 where it contacts the spores, the nucleic acid interacting dye and/or the enhancer reagent.
  • the sterilant gas inactivates the spores, the nucleic acid-interacting dye can penetrate the spore and interact with the nucleic acid therein.
  • the container is opened (e.g., by crushing) prior to exposing the sterilization process indicator to the sterilant gas.
  • the container is opened (e.g., by crushing) after exposing the sterilization process indicator to the sterilant gas. Opening the container places the spores in fluid contact with the aqueous liquid medium containing the nutrient composition.
  • a self-contained biological indicator of the present disclosure can comprise a nucleic acid-interacting dye that, when bound to a nucleic acid can cause a change in the electrical conductance (or resistivity) of an aqueous mixture in which the dye and the nucleic acid are disposed. In some embodiments, this change can be due to a redox reaction in which the nucleic acid- bound dye can participate. Thus, the presence or absence of the dye bound to nucleic acid can be detected electrochemically (e.g., by measuring conductance of the aqueous medium.
  • a nucleic acid-interacting dye is Hoechst 33258 DNA binding dye (available from VWR International, Radnor PA).
  • the present disclosure additionally provides a method.
  • the method can be used to determine the effectiveness of a sterilization process.
  • the method includes positioning any embodiment of a self-contained sterilization process biological indicator according to the present invention in a sterilization chamber.
  • the sterilization chamber can be a sterilization chamber of a sterilizer (e.g., a commercially-available automated sterilizer), the sterilization chamber being typically sized to contain a plurality of articles to be sterilized and equipped with a means of evacuating air and/or other gases from the chamber and adding a sterilant gas to the chamber.
  • the biological sterilization indicator can be positioned in the most difficult location (e.g., above the drain) in the sterilizer to achieve proper sterilization conditions (e.g., temperature, pressure).
  • the biological sterilization indicator can be positioned adjacent an article to be sterilized when placed in the sterilization chamber.
  • the biological sterilization indicator can be adapted into routinely-used process challenge devices before positioning it in the stenlization chamber.
  • the method includes a step of exposing the sterilization process indicator to a sterilant gas (e.g., during a sterilization process).
  • the sterilization process indicator is exposed to the sterilant gas in the sterilization chamber.
  • exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to steam.
  • exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to ethylene oxide.
  • exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to a peroxide.
  • exposing the sterilization process indicator to a sterilant gas comprises exposing the sterilization process indicator to ozone.
  • a sterilization process indicator comprises an opening that is part of a pathway that permits passage of a sterilant gas from outside the cuvette into the chamber of the indicator; exposing the sterilization process indicator to the sterilant gas comprises exposing the spores disposed in the compartment of the indicator to the sterilant gas.
  • exposing the sterilization process indicator to the sterilant gas comprises exposing the spores to the sterilant gas under conditions (e.g., time, temperature, pressure, and concentration of sterilant gas) selected to be sufficient to inactivate (e.g., render nonviable and/or non- cultivable) all of the spores of the predetermined number of sterilization process-resistant spores disposed in the sterilization process indicator.
  • conditions e.g., time, temperature, pressure, and concentration of sterilant gas
  • the sterilant gas can be added to the sterilization chamber after evacuating the chamber of at least a portion of any air or other gas present in the chamber.
  • the sterilant gas may be added to the sterilization chamber without evacuating the chamber.
  • a series of evacuation steps is often used to assure that the sterilant gas reaches all areas within the sterilization chamber and contacts all areas of the article(s) in the sterilization chamber to be sterilized.
  • the sterilant gas also contacts the spores under conditions (e.g., temperature, pressure, concentration) where the sterilant gas reaches all areas within the sterilization chamber.
  • the method of the present disclosure further comprises contacting the spores with the nucleic acid-interacting dye.
  • the spores are contacted with the nucleic acid-interacting dye in the compartment of the sterilization process indicator.
  • contacting the spores with the nucleic acid-interacting dye comprises contacting the spores with the nucleic acid-interacting dye while exposing the sterilization process indicator to the sterilant gas.
  • the container is opened (e.g., by fracturing, crushing or otherwise disintegrating the container) prior to the exposing the indicator to the sterilant gas (e.g., before the indicator is positioned in the stenlization chamber).
  • the method of the present disclosure further comprises contacting the spores with the liquid medium.
  • the self-contained sterilization process biological indicator comprises a container disposed in the compartment, wherein the container contains the liquid medium.
  • the method further comprises disintegrating the container to contact the spores with the aqueous liquid medium.
  • the spores are not in contact with the aqueous liquid medium while the spores are exposed to the sterilant gas.
  • the method of the present disclosure further comprises contacting the spores with the nutrient composition.
  • the method further comprises disintegrating the container to contact the spores with the aqueous nutrient composition.
  • the method comprises incubating the sterilization process indicator at a predetermined temperature for a period of time sufficient to permit germination and at least one cell division of a germinated spore.
  • an incubation temperature above room temperature may be used.
  • the incubation temperature is at least 37° C.
  • the incubation temperature is at least 50° C.
  • the incubation temperature is 50 to 60° C.
  • the sterilization process indicator can be incubated for a period of time sufficient to permit germination and at least one cell division of a germinated spore.
  • the incubation can be not less than 15 minutes, not less than 30 minutes, not less than 1 hour, not less than 2 hours, not less than 4 hours, not less than 6 hours, not less than 8 hours, or not less than 12 hours.
  • the incubation can be up to 30 minutes, up to 1 hour, up to 2 hours, up to 4 hours, up to 6 hours, up to 8 hours, up to 12 hours, or up to 24 hours.
  • the method comprises measuring a parameter associated with a germinated spore.
  • Parameters associated with a germinated spore include, but are not limited to, metabolic activity (e g., catabolic consumption of nutrients to produce cellular energy, anabolic production of biomolecules, production of metabolic byproducts (e.g., CO2, organic acids, alcohols), and enzyme activities found in germinated spores and microbial vegetative cells.
  • measuring the parameter associated with a germinated spore comprises measuring a second amount of absorbance and/or scattering of an electromagnetic radiation as the electromagnetic radiation is directed through the nutrient composition.
  • Measurement of the second amount of absorbance or light-scattering is generally related to particles (e.g., germinated spores and vegetative cells) in the liquid medium of the sterilization process indicator. Spores that have survived the exposure to the sterilant gas are able to germinate and reproduce using the nutrient composition in the sterilization process indicator, thereby increasing the absorbance and/or scattering of light directed through the sterilization process indicator.
  • the measured absorbance/light-scattering value is greater than a predetermined value (e.g., an arbitrary threshold or a value measured in a“control” sterilization process indicator in which all of the spores have been killed), it can be concluded that at least one spore in the sterilization process indicator survived the exposure to the sterilant gas.
  • a predetermined value e.g., an arbitrary threshold or a value measured in a“control” sterilization process indicator in which all of the spores have been killed
  • Measuring a first or second amount of absorbance and/or scattering of an electromagnetic radiation as the electromagnetic radiation is directed through the nutrient composition can be done using any wavelength of electromagnetic radiation typically used to detect turbidity that is due to microorganisms.
  • the wavelength can be about 420 nm, 600 nm, or 660 nm. In certain preferred embodiments, the wavelength is about 420 nm to about 660 nm. In certain preferred embodiments, measuring the amount of absorbance and/or scattering of an electromagnetic radiation as the electromagnetic radiation that is directed through the aqueous liquid medium can be performed as described hereinabove
  • the second amount of absorbance or light-scattering can be monitored and measured continuously or intermittently while incubating the spores with the liquid medium comprising the nutrient composition.
  • detecting the parameter associated with a germinated spore comprises detecting and/or measuring an enzyme activity associated with a germinated spore.
  • Suitable enzyme activities to detect germinated spores of Geobacillus stearothermophilus include, but are not limited to, alpha-D-glucosidase, beta-D-glucosidase, alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase, lipase, leucine aminopeptidase, chymotrypsin, phosphohydrolase, alpha-D-galactosidase, beta-D-galactosidase, alanine
  • Suitable enzyme activities to detect Bacillus atrophaeus include, but are not limited to, alpha-L- arabinofuranosidase, beta-D-glucosidase, N-acetyl-p-glucosaminidase, beta-D-cellibiosidase, alanine aminopeptidase, leucine aminopeptidase, and phenylalanine aminopeptidase.
  • the sterilization process monitor of the present disclosure comprises a detection reagent comprising a fluorogenic or chromogenic enzyme substrate, as described in U.S. Patent No. 6, 623,955, entitled “RAPID READ-OUT BIOLOGICAL INDICATOR”, which is incorporated herein by reference in its entirety.
  • detecting the parameter associated with a germinated spore comprises detecting and/or measuring a product of microbial metabolism.
  • the product of microbial metabolism can be, for example, an organic acid produced by incomplete oxidation of one of the components of the nutrient composition.
  • the accumulation of the product in the liquid medium can be detected, for example, by including a detection reagent such as a pH indicator (e.g., bromcresol purple) into the sterilization process monitor.
  • a detection reagent such as a pH indicator (e.g., bromcresol purple) into the sterilization process monitor.
  • the pH indicator can be incorporated into the liquid medium or the nutrient composition.
  • detecting a parameter associated with a germinated spore comprises detecting a change in a pH indicator from a first state (e.g., first color) to a second state (e.g., second color) in the liquid medium of the sterilization process indicator.
  • the present method which uses the process indicators described above, can, therefore, be sufficiently sensitive to the presence of viable spores to provide an indication thereof in a short period of time.
  • the indication can be provided even when the number of viable spores present is relatively low.
  • the rapid indication of spore viability e.g., as evidenced by a first fluorescent intensity that is higher than the reference fluorescence intensity
  • the method of the present disclosure comprises measuring a first fluorescence intensity emitted by the nucleic acid-interacting dye in the self-contained sterilization process biological indicator. Measuring the first fluorescence intensity is performed when the spores, the liquid medium, and the nucleic acid-interacting dye are all in fluid communication.
  • the container is opened as described herein before the first fluorescence intensity is measured. Opening the container places the spores in contact with the nutrient composition disposed in the container.
  • Measuring the first fluorescence intensity can comprise illuminating at least a portion of the cuvette with electromagnetic radiation of a first (excitation) wavelength and intensity sufficient to cause the nucleic acid interacting dye that is bound to nucleic acid to fluoresce at a second (emission) wavelength.
  • the first fluorescence intensity is measured (e.g., in a fluorometer adapted to receive the cuvette) at the second wavelength and is an indication of the effectiveness of the inactivation of the spores in the sterilization process indicator, as discussed hereinabove.
  • the method After the measuring the first fluorescence intensity, the method also comprises comparing the first fluorescence intensity to a reference fluorescence intensity to determine whether the exposing the sterilization process biological indicator to the sterilant gas was effective to kill all of the spores.
  • the reference fluorescence intensity is a measured intensity of fluorescence (i.e., a second fluorescence intensity) emitted by a“control” sterilization process indicator (e.g., of a“positive control”, such as an identical sterilization process indicator that has not been exposed to a sterilant gas and, thus, the spores are substantially all viable; or a“negative control”, such as an identical sterilization process indicator that has been treated (e.g., in an “overkill” sterilization process) so that substantially all of the spores are nonviable).
  • a“control” sterilization process indicator e.g., of a“positive control”, such as an identical sterilization process indicator that has not been exposed to a sterilant gas and, thus, the spores are substantially all viable
  • a“negative control” such as an identical sterilization process indicator that has been treated (e.g., in an “overkill” sterilization process) so that substantially all of the spores are nonviable).
  • the reference fluorescence intensity is a measured intensity (i.e., a second fluorescence intensity) emitted by a“fluorescence control” (e.g., a solution or a solid state material that emits a predetermined fluorescence intensity used to set a threshold intensity value to which the first fluorescence intensity can be compared to determine whether or not all of the spores in the sterilization process monitor that was exposed to the sterilant gas have been inactivated.
  • a“fluorescence control” e.g., a solution or a solid state material that emits a predetermined fluorescence intensity used to set a threshold intensity value to which the first fluorescence intensity can be compared to determine whether or not all of the spores in the sterilization process monitor that was exposed to the sterilant gas have been inactivated.
  • the reference fluorescence intensity can be an arbitrary fluorescence intensity value, for example, in a printed publication such as instructions for use that can be used by the operator to make a comparison with the first fluorescence intensity, or in an electronic data set that can be used (e.g., by a fluorimeter or a microprocessor) to make a comparison with the first fluorescence intensity.
  • Comparing the first fluorescence intensity to a reference fluorescence intensity comprises determining whether the first fluorescence intensity is less than, equal to, or greater than the reference fluorescence intensity. In certain embodiments, a first fluorescence intensity that is greater than the reference intensity indicates the sterilization process was effective to inactivate all of the
  • a first fluorescence intensity that is greater than or equal to the reference intensity indicates the sterilization process was effective to inactivate all of the predetermined number of spores. In certain embodiments, a first fluorescence intensity that is less than the reference intensity indicates the sterilization process was not effective to inactivate all of the predetermined number of spores. In certain embodiments, a first fluorescence intensity that is less than or equal to the reference intensity indicates the sterilization process was not effective to inactivate all of the predetermined number of spores.
  • a method according to the present disclosure optionally can comprise measuring a first amount of absorbance and/or scattering of electromagnetic radiation when the electromagnetic radiation is directed through the aqueous liquid medium in the cuvette.
  • the first amount absorbance and/or scattering is measured after the exposing the sterilization process indicator to the sterilant gas.
  • the wavelength of electromagnetic radiation used to measure the absorbance and/or scattering can be, for example, a wavelength used in the art to assess the optical density of spores suspended in a liquid medium.
  • suitable wavelengths for measuring the absorbance and/or scattering are those wavelengths in the range of about 500nm to about 600nm.
  • the method further includes a step of calculating a ratio of the first fluorescence intensity to the measured amount of electromagnetic radiation absorbance and/or scattering.
  • measuring the amount of absorbance and/or scattering of an electromagnetic radiation as the electromagnetic radiation that is directed through the aqueous liquid medium can be performed using a device (e.g., a spectrophotometer, a colorimeter) capable of receiving the sterilization process indicator and measuring the absorbance and/or light-scattering of the liquid contents therein.
  • a device e.g., a spectrophotometer, a colorimeter
  • the ratio of the first fluorescence intensity to the measured first amount of electromagnetic radiation absorbance and/or scattering can be used to calculate and adjusted first fluorescent intensity (IA) according to the following formula:
  • the method further can comprise agitating the self-contained sterilization process biological indicator for a period of time. Agitation can be used to mix the contents present in the chamber of the indicator. The indicator can be agitated, for example, to suspend the spores in the liquid medium (e.g., after a container has been actuated to release the liquid medium into the chamber).
  • the indicator can be agitated before measuring the first fluorescence intensity and/or before measuring the amount of absorbance and/or scattering of electromagnetic radiation as the electromagnetic radiation is directed through the aqueous liquid medium in the cuvette.
  • the indicator can be agitated manually (e.g., by swirling, tapping, or shaking) or it can be agitated using an sample-vortexing machine.
  • the method of the present disclosure comprises contacting the spores with the nutrient composition.
  • Contacting the spores with the nutrient composition comprises opening the container (containing the nutrient composition in dry form or in the liquid medium) to place the spores in fluid communication with the nutrient composition.
  • the container can be opened as discussed hereinabove.
  • the method further comprises placing an article to be sterilized along with the sterilization process indicator in the sterilization chamber.
  • the method further comprises determining whether or not the sterilization process was effective for sterilizing the article. An indication of no viable spores may be used to determine that the sterilization process was effective for sterilizing the article, whereas an indication of viable spores may be used to determine that the process was not effective.
  • an assessment of the sterility of an article subjected to a sterilization process may be made in a relatively short time using the indicator and method embodiments described above by directly measuring production of nucleic acids m any viable spores.
  • detection of the nucleic acid-interacting dye with a nucleic acid after a sterilization process can be effected by measuring the electrical conductance of a liquid medium in which the nucleic acid interacting dye with the nucleic acid, as described, for example, in Examples 7 and 8 herein.
  • Example 1 Assembly of a Self-Contained Sterilization Process Biological Indicator.
  • Spores of Geobacillus stearothermophilus can be produced in liquid sporulation medium.
  • the spores can be washed in sterile deionized water and resuspended in sterile deionized water to achieve a concentration of about 10 8 -10 9 spores/mL.
  • Bio indicators (part number 1292) can be obtained from 3M Company (St. Paul, MN). Caps on the biological indicators can be removed and the contents (glass ampule, spore strip, and nonwoven packing adjacent the spore strip in the bottom of each tube) can be removed. The inside of the tubes and the outside of the glass ampules can be rinsed with sterile deionized water.
  • each tube can be left in place.
  • the caps (with the nonwoven sheets therein) can be replaced on the tubes and each assembled tube can be sterilized in a steam sterilizer.
  • the spore suspension can be diluted in a sterile solution of SYTO 9 dye (Invitrogen, Carlsbad, CA) to obtain a final spore suspension having about 2 c lOVmL in a solution of 40 mM SYT09 dye.
  • SYTO 9 dye Invitrogen, Carlsbad, CA
  • Ten-microliter aliquots of the resulting spore suspension can be pipetted onto polyethylene film discs (about 6 mM diameter).
  • the spore-coated discs can be dried at 50 degrees C for about 30 minutes.
  • the self-contained sterilization process indicators can be assembled by removing the caps from the sterilized tubes, placing one of the spore-coated discs and one of the washed glass ampules into the bottom each of the sterilized tubes, and replacing the caps on the tubes.
  • Geobacillus stearothermophilus ATCC 7953 spores were suspended in sterile H2O (3.4xl0 6 spores/mL) and aliquoted (2 mL volumes) into six glass test tubes.
  • Table 9 shows the six different steam -exposure times at 121°C in a dynamic-air removal steam sterilization process in a MidMark M9 sterilizer.
  • Commercial 1492V biological indicators (3M Company, St. Paul, MN) were used as growth controls as described below.
  • Table 2 Reference Example 1 Data.
  • the OD600, Syto9 (RFU), and Syto9 (RFU)/OD600 reported in this table are the average of the triplicate technical replicates for each condition.
  • the CFU/mL is the average of duplicate technical replicates for each condition.
  • Example #2 Dried spores - DNA dye added to spore coating solution
  • Geobacillus stearothermophilus ATCC 7953 spores were suspended in an aqueous trehalose solution (25% w/v) with and without syto9 dye (10mM) and spotted (20pF) onto circular polypropylene film carriers to yield approximately >10 6 spores/carrier.
  • the coated carriers were then dried at 60°C for 15 minutes.
  • the spore-coated and dried carriers were then exposed to a 6-minute steam sterilization process at 121°C in a dynamic-air removal steam sterilization cycle using a MidMark M9 sterilizer. Samples were prepared and tested in duplicate.
  • the data for this example are shown in Table 3, 4 and 5.
  • the exposure time indicates the length of exposure to steam at 121C in a Midmark M9 commercial sterilizer.
  • Syto9 pre/post sterilization indicates whether the sample had syto9 dye added to the coating solution (Pre- sterilization), or after the sterilization process (Post).
  • the RFU/OD600 values shown in Table 3 correlated well with the length of exposure of the spores to the steam sterilization process. The observed fluorescent signal in the steam-exposed samples was higher when the dye is added prior to sterilization.
  • Example #3 Dried spores in biological indicator
  • Geobacillus stearothermophilus ATCC 7953 spores were suspended in a trehalose solution (25% w/v) with syto9 dye (IOmM) and spotted (20pL) on the inside of the wall of polycarbonate housings from 3MTM ATTESTTM 1295 biological indicators. Each resulting biological indicator contained approximately >10 6 spores coated on inside of the wall of the housing.
  • the coated BI sleeves were then dried at 56°C for approximately 1 hour.
  • SRBI caps were then placed on the coated BI sleeves.
  • the Samples were then exposed to steam at 121°C in a dynamic-air-removal benchtop commercial sterilization vessel at 2 different exposure points (TO exposure and 6 minutes exposure). Samples were prepared in duplicate.
  • EbO (220 pL) was added to each BI.
  • the Bis were then vortexed for 3 seconds. Aliquots were then pulled (200 pL) from each BI and the fluorescence and optical density were immediately measured in a BIOTEK 96-well plate reader at room temperature as described in Reference Example 3.
  • the spore pellet in the microcentrifuge tube was then resuspended with the coating solution (1 mL).
  • Four pi of Syto9 (5000 mM stock) was then added to the suspension to yield a 20 mM concentration.
  • the suspension was then coated onto polypropylene film carriers. Carriers were dried for 25 minutes at 56°C.
  • the biological indicators were then placed in an H&W 105 resistometer, where they were exposed to an ISO 132°C Pre-vacuum sterilization cycle for various periods of 132°C exposure ( 30 seconds, 1.5 minutes, 2 minutes, 2.5 minutes, and 3.5 minutes).
  • sterile EbO (220 pL) was added to each Biological indicator and the indicators were vortexed briefly to resuspend spores. The resuspended spores were then transferred to a clear bottom, black walled 96-well plate. Plates were read at room temperature in the BioTek plate reader at 480nm excitation/520nm emission, and the OD600 was measured. Readings were performed as quickly as possible from the time of resuspension to reading in the plate reader. Following the fluorescence reading, nutrient media (containing bromocresol purple) that supports spore germination and outgrowth was added to the wells. The plate was then placed in a 60°C incubator for a minimum of 24 hours, after which the media was examined for indications (e.g., turbidity, pH change) of spore germination and growth.
  • indications e.g., turbidity, pH change
  • Table 7 and 8 show the fluorescence and growth data respectively for this example.
  • the values shown in Table 7 are relative fluorescence units (RFU) for five biological indicator replicates across 5 exposure times.
  • the RFU values shown in Table 7 are with the background (from unexposed BI’s) subtracted.
  • Table 8 shows the growth result with bromocresol purple and media that supports spore germination and outgrowth.
  • a threshold value could be set that indicates where positive and negative fluorescence could be established based on how it correlates to spore growth. For example anything below 8000 RFU would be assigned a fluorescent positive value (Growth positive) and anything above 8000 RFU would be assigned a fluorescent negative value (Growth negative).
  • This data set indicates an example reference standard curve data set that could be used to assign fluorescent designation for near immediate monitoring of sterilization cycles.
  • AttestTM1295 biological indicators were placed into a TyvekTM polyethylene fiber pouch that was exposed to H2O2 vapor for 90 seconds of exposure in an otherwise empty Sterrad® 100NX sterilizer. Preliminary experiments showed that a 90-second exposure in the sterilizer was sufficient to kill all of the spores in the biological indicators.
  • the carriers were then removed from 3 of the ThOa-exposed biological indicators and 3 unexposed (control) biological indicators and the carriers were placed in separate borosilicate glass vials with 1 mL of Butterfields buffer. The vials were then vortexed briefly and sonicated for 15 minutes in a water bath sonicator.
  • Sample preparation [00156] A sample stock solution of DNA (Lambda DNA available from Invitrogen, Waltham, MA) is prepared in a 50 mM phosphate buffer (40 mM K2HPO4 and 10 mM KH2PO4, pH 7.0) to a DNA concentration of 100 pg/mL. The stock solution is diluted with additional buffer to make a range of sample solutions with DNA concentrations of: 10, 1 and 0.1 pg/mL. A control sample solution containing no DNA is also prepared. The DNA sample solutions simulate samples of spore DNA that is released as a result of a sterilization process. The varying concentrations of DNA in the sample solutions simulate exposures to the sterilant for different exposure times.
  • Hoechst 33258 DNA binding dye (available from VWR International, Radnor PA) is added to the DNA sample solutions as well as the control solution, to give a concentration of 1 pM for the Hoechst binding dye.
  • the Hoechst binding dye concentration is constant (1 pM) for all DNA sample solutions as well as the control solution.
  • LSV oxidative linear sweep voltammetry
  • the electrochemical cell consists of a Metrohm DropSense screen printed three electrode cell (DropSense 220AT available from Metrohm, Riverview, FL).
  • the electrochemical cell has gold working and auxiliary electrodes and a silver reference electrode.
  • the working electrode is 4mm in diameter.
  • the DNA sample solutions are tested by placing a 100 pL drop of the sample solution onto the screen-printed electrochemical cell covering all three electrodes. After a five-minute equilibration at 0 volts, the LSV described above is initiated. The LSV analysis is repeated for the control solution containing no DNA. For each LSV analysis a new electrochemical cell is used.
  • the LSV curves will show oxidation of the Hoechst binding dye at 0.6V.
  • the peak current measured in pA at this oxidation potential is always lower for the control solution in comparison to the DNA sample solutions.
  • the ratio of the peak current at the oxidation potential for the series of DNA sample solution divided by the peak current of the control solution at that same potential increases proportionally with increasing DNA concentration in the sample solution series.
  • Hoechst 33258 (available from VWR International, Radnor, PA) nucleic acid binding dye is added to aliquots of each test sample and LSV is immediately conducted. LSV is performed using a Metrohm AUTOLAB potentiostat (available from Metrohm, Riverview, FL) in the voltage range from 0 to 1.0 V at a sweep rate of 100 mV/s.
  • the electrochemical cell consists of a Metrohm
  • DropSens screen printed three electrode cell (DropSense 220AT available from Metrohm, Riverview, FL).
  • the electrochemical cell has gold working and auxiliary electrodes and a silver reference electrode.
  • the working electrode is 4mm in diameter.
  • the DNA sample solutions are tested by placing a 100pL drop of the test solution onto the screen-printed electrochemical cell covering all three electrodes. After a five-minute equilibration at 0 volts, the LSV described above is initiated.
  • the colony forming units for each test sample was determined by diluting the spore suspensions and surface-plating the dilutions onto Tryptic Soy Agar followed by incubation of the plates at 60°C.
  • the LSV curves show oxidation of the Hoechst binding dye at 0.6V.
  • Sample 6 results the lowest peak current at the oxidation potential.
  • Sample 1 results in a peak current greater than sample 6 at the oxidation potential. This is because, even though the sterilization exposure is TO (meaning the cycle is stopped at the very beginning of the sterilization exposure phase of the cycle) the preconditioning portion of the cycle results in a very small amount of released DNA available for detection.
  • the peak current for samples 2-5 is significantly larger than for sample 6, indicating greater amounts of DNA detected electrochemically.
  • the peak current for samples 4 and 5 is comparable, indicating that for exposure times greater than 6 minutes all the possible DNA generated as a result of the sterilization cycle is detected electrochemically. 1492V commercial biological indicator controls are all growth negative at 2 minutes of exposure in this experiment.
  • Sample Preparation [00170] Sample Preparation [00171] Commercial 3MTM AttestTM1295 biological indicators (available from 3M, St. Paul, MN) are exposed to VH202 in an incomplete Standard Cycle for the Sterrad 100NX (available from ASP, Irvine, CA) sterilizer providing a total of 90 seconds of exposure. Six of the BI's from this lot are packaged in a TYVEK® polypropylene fiber pouch and placed in an otherwise empty chamber. This cycle is run in order to kill the BI's.
  • the carriers are then removed from 3 of the exposed (Dead) and unexposed (Live) BI's and placed in separate borosilicate glass vials with 1 mL of Butterfields buffer. The vials are vortexed briefly and sonicated for 15 minutes in a water bath sonicator.
  • 100pL of each sample is placed in a microcentrifuge tube. To each sample, 100pL of a
  • a control sample with no Hoechts dye added is prepared for both Dead and Live samples. Samples are mixed and immediately tested using LSY. LSV is performed using a Metrohm AUTOLAB potentiostat (available from Metrohm, Riverview, FL) in the voltage range from 0 to 1.0 V at a sweep rate of 100 mV/s.
  • the electrochemical cell consists of a Metrohm DropSens screen printed three electrode cell (DropSense 220AT available from Metrohm, Riverview, FL). The electrochemical cell has gold working and auxiliary electrodes and a silver reference electrode. The working electrode is 4mm in diameter.
  • the DNA sample solutions are tested by placing a 100pL drop of the test solution onto the screen-printed electrochemical cell covering all three electrodes. After a five-minute equilibration at 0 volts, the LSV described above is initiated.

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Abstract

L'invention concerne un indicateur biologique de processus de stérilisation autonome. L'indicateur comprend une cuvette ayant au moins une paroi imperméable aux liquides formant une ouverture dans un compartiment, un colorant interagissant avec l'acide nucléique disposé dans le compartiment, un nombre prédéterminé de spores résistantes au processus de stérilisation disposées dans le compartiment, un premier milieu liquide disposé dans le compartiment, et une composition nutritive facilitant la germination et/ou l'excroissance d'une spore viable. Les spores dans l'indicateur biologique de processus de stérilisation autonome sont en contact avec le colorant. La composition nutritive est disposée dans le compartiment et isolée des spores. L'ouverture fait partie d'un trajet qui permet le passage d'un gaz stérilisant dans le compartiment. Le colorant interagissant avec l'acide nucléique est fluorescent lorsqu'il interagit avec de l'ADN ou de l'ARN. L'invention concerne également un procédé d'utilisation de l'indicateur biologique de processus de stérilisation autonome pour déterminer l'efficacité d'un processus de stérilisation.
PCT/IB2019/061398 2018-12-27 2019-12-27 Indicateur biologique de lecture instantanée avec confirmation de croissance WO2020136613A1 (fr)

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Publication number Priority date Publication date Assignee Title
US5073488A (en) 1988-11-29 1991-12-17 Minnesota Mining And Manufacturing Company Rapid method for determining efficacy of a sterilization cycle and rapid read-out biological indicator
WO1999005310A1 (fr) 1997-07-28 1999-02-04 Minnesota Mining And Manufacturing Company Milieu de germination de spores
US20020123089A1 (en) * 1999-05-03 2002-09-05 Icf Technologies, Inc. System for detecting sterilization effectiveness
US6623955B2 (en) 1988-11-29 2003-09-23 3M Innovative Properties Company Rapid read-out biological indicator
US20070238145A1 (en) * 2006-02-21 2007-10-11 Cote Mindy A Phenotypic engineering of spores
WO2010045138A2 (fr) 2008-10-17 2010-04-22 3M Innovative Properties Company Indicateur de stérilisation biologique, système associé et ses procédés d'utilisation
US20160186120A1 (en) * 2010-11-01 2016-06-30 3M Innovative Properties Company Biological sterilization indicator
US20170218428A1 (en) * 2013-05-21 2017-08-03 3M Innovative Properties Company Nanostructured spore carrier
US20170292143A1 (en) * 2006-02-21 2017-10-12 Bcr Diagnostics, Inc. Phenotypic engineering of spores

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Publication number Priority date Publication date Assignee Title
US5073488A (en) 1988-11-29 1991-12-17 Minnesota Mining And Manufacturing Company Rapid method for determining efficacy of a sterilization cycle and rapid read-out biological indicator
US6623955B2 (en) 1988-11-29 2003-09-23 3M Innovative Properties Company Rapid read-out biological indicator
WO1999005310A1 (fr) 1997-07-28 1999-02-04 Minnesota Mining And Manufacturing Company Milieu de germination de spores
US20020123089A1 (en) * 1999-05-03 2002-09-05 Icf Technologies, Inc. System for detecting sterilization effectiveness
US20070238145A1 (en) * 2006-02-21 2007-10-11 Cote Mindy A Phenotypic engineering of spores
US20170292143A1 (en) * 2006-02-21 2017-10-12 Bcr Diagnostics, Inc. Phenotypic engineering of spores
WO2010045138A2 (fr) 2008-10-17 2010-04-22 3M Innovative Properties Company Indicateur de stérilisation biologique, système associé et ses procédés d'utilisation
US20110182770A1 (en) * 2008-10-17 2011-07-28 Sailaja Chandrapati Biological sterilization indicator, system, and methods of using same
US20150165082A1 (en) 2008-10-17 2015-06-18 3M Innovative Properties Company Biological sterilization indicator, system, and methods of using same
US20160186120A1 (en) * 2010-11-01 2016-06-30 3M Innovative Properties Company Biological sterilization indicator
US10047334B2 (en) 2010-11-01 2018-08-14 3M Innovative Properties Company Biological sterilization indicator
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MACLNNES, J. WMCCLINTOCK, M.: "Differences in Fluorescence Spectra of Acridine Orange-DNA Complexes Related to DNA Base Composition", BIOPOLYMERS. COMMUNICATIONS TO THE EDITOR, vol. 9, 1970, pages 1407 - 1411
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