Biological indicator comprising a genetically modified test microorganism

Abstract

The invention provides an article for assessing the efficacy of a sterilization process. The article includes a housing, a plurality of genetically modified spore-forming test microorganisms disposed in the housing, a liquid medium disposed in an openable container, and an enzyme substrate disposed in the housing or the openable container, wherein the openable container is disposed in or attached to the housing. The genetically modified test microorganism comprises a functional fusion gene encoding a non-naturally occurring chimeric protein comprising a first segment and a second segment contiguous with the first segment. The first segment comprises at least a portion of a first polypeptide that normally occurs in a spore, and the second segment comprises a second polypeptide having a detectable enzymatic activity. The enzyme substrate is capable of reacting with the detectable enzymatic activity to form a detectable product. The invention also provides a method of using the test microorganism.

Description

[0001] sequence list

[0002] This application contains a sequence list, submitted electronically to the International Bureau as ASCII text via ePCT, which is 8.65 kilobytes in size, named "82825US002_SEQUENCE_LISTING_ST25.txt", and created on September 2, 2020. Information contained in the sequence list is incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to sterilization indicators, and more particularly to biological sterilization indicators and methods for assessing the effectiveness of sterilization processes. Background Technology

[0004] Sterilization of equipment, instruments, and other devices is crucial in the healthcare industry. For example, hospitals and other healthcare facilities frequently sterilize medical devices and equipment used in the treatment of patients. The specific type of sterilization cycle used to sterilize such equipment can vary based on the particular equipment or device to be sterilized and the specific preferences of the entity performing the sterilization cycle. However, all such sterilization cycles or processes are generally designed to kill any living organisms that might otherwise contaminate the equipment or device to be sterilized.

[0005] Various sterilization methods utilize different cycles or techniques to sterilize. For example, sterilization may include applying steam, dry heat, chemicals (e.g., ethylene oxide), or radiation to the equipment or apparatus to be sterilized. Steam sterilization is generally effective when the equipment to be sterilized is heat-resistant at high temperatures, as these items are exposed to steam at temperatures generally in the range of 121°C–135°C. The duration of steam exposure depends on the sterilization temperature. For example, equipment or instruments to be sterilized are preferably exposed to steam at 132°C for approximately three minutes. However, exposure times at 121°C can be up to 30 minutes.

[0006] Bioindicators are commonly used to evaluate and validate the effectiveness of sterilization processes in various environments. Generally, they involve subjecting live, but relatively resistant, spores (test microorganisms) of thermophilic organisms, along with any device or instrument to be sterilized, to sterilization conditions. Generally, spores are more resistant to sterilization processes than most other organisms that would otherwise be susceptible to natural contamination. Applications utilize spores from microorganisms capable of producing enzymes that catalyze the reaction of a non-fluorescent substrate into a fluorescent product, which can be detected to indicate the presence of viable spores.

[0007] Typically, after the sterilization process is complete, the test microorganisms (e.g., spores) are incubated in a liquid culture medium containing indicator compounds to determine whether any of the test organisms survived the sterilization procedure. In conventional biological indicators, when using pH indicators to detect growth, contacting the nutrient composition with the test microorganisms and observing the growth of a detectable number of test microorganisms can take up to 24 hours.

[0008] Using rapid readout techniques to detect the activity of enzymes associated with test microorganisms can reduce the time required to detect live test microorganisms. In some embodiments, analysis of the fluorescence intensity caused by the fluorescent products of the enzyme reaction is used to determine whether the sterilization process was successful. Summary of the Invention

[0009] A complete set of sterilization process bioindicators is now available, providing everything needed to rapidly assess the effectiveness of various steam sterilization processes by detecting the germination and / or growth of live, genetically modified test microorganisms (if present) after exposure of the complete set of sterilization process bioindicators to the sterilization process. Advantageously, this discovery provides its users with bioindicators containing genetically modified test microorganisms that are not naturally occurring and can be rapidly detected after the sterilization process. Surprisingly, the engineered microorganisms of this disclosure not only reduce the time required to detect spore viability after sterilization, but also exhibit chimeric protein enzymatic activity that better correlates with the spore growth response after exposure to the sterilization process.

[0010] This disclosure provides genetically modified test microorganisms that produce a chimeric protein containing detectable enzyme activity. Advantageously, the chimeric protein comprises a polypeptide that accumulates in high copy numbers in the spores of the test microorganism during spore formation. Furthermore, the fusion gene encoding the chimeric protein increases the amount of enzyme activity in the spores. This feature allows for faster detection of enzyme activity than in unmodified microorganisms.

[0011] In one aspect, this disclosure provides an article of manufacture for evaluating the effectiveness of a sterilization process. The article of manufacture may include a shell, a plurality of genetically modified test microorganisms disposed within the shell, a liquid culture medium disposed within an openable container, and an enzyme substrate disposed within or attached to the shell. Each of the plurality of genetically modified test microorganisms comprises a sporulating genetically modified test microorganism. The genetically modified test microorganisms comprise a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment. The first segment may comprise at least a portion of a first polypeptide normally present in spores. The second segment may comprise a second polypeptide having detectable enzyme activity. The enzyme substrate is capable of reacting with the detectable enzyme activity to form a detectable product.

[0012] In any of the above embodiments of the in-process product and the embodiments of the integrated sterilization process bioindicator, the first polypeptide may be selected from small acid-soluble spore proteins, spore coat proteins, spore formation-related GTP-binding proteins, and spore germination proteins. In some embodiments, the first polypeptide is encoded by a gene selected from ysxE, yutH, cotE, cotF, cotH, cotS, cotF, gerE, sspA, sspB, sspD, and sspE. In any of the above embodiments, the second polypeptide has enzymatic activity selected from esterases, lipases, glycosidases, aminopeptidases, phosphatases, and luciferases. In any of the above embodiments, the first segment comprises the N-terminal region of the first polypeptide. In any of the above embodiments, at least a portion of the first polypeptide normally present in spores comprises a first polypeptide segment comprising not less than 1% of the amino acid residues constituting the first polypeptide.

[0013] In another aspect, this disclosure provides a method for determining the effectiveness of a sterilization process. The method may include a means of determining the effectiveness of a sterilization process. A bioindicator is present on or contains a plurality of genetically modified test microorganisms, each of the plurality of genetically modified test microorganisms comprising a sporulating genetically modified test microorganism; wherein the genetically modified test microorganism contains a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment; wherein the first segment comprises at least a portion of a first polypeptide normally present in spores; wherein the second segment comprises a second polypeptide having detectable enzyme activity. The method may further include exposing the bioindicator to a sterilizing agent gas while positioning the indicator in a sterilization chamber; after exposing the bioindicator to the sterilizing agent gas, contacting the test microorganism with a liquid culture medium and an enzyme substrate capable of reacting with enzyme activity to form a detectable product; after contacting the genetically modified test microorganism with the liquid culture medium and the enzyme substrate, incubating the bioindicator at a predetermined temperature for a period of time; and detecting the product in the liquid culture medium.

[0014] In some embodiments of the above method, positioning a sterilization process bioindicator in a sterilization chamber includes positioning a sterilization process bioindicator comprising a shell having a test microorganism therein, wherein contacting the test microorganism with a liquid culture medium includes contacting the test microorganism with a liquid culture medium inside the shell.

[0015] In another aspect, this disclosure provides a kit. The kit may comprise a plurality of genetically modified test microorganisms, each of which comprises a sporulating genetically modified test microorganism; wherein the genetically modified test microorganism comprises a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment; wherein the first segment comprises at least a portion of a first polypeptide normally present in spores; wherein the second segment comprises a second polypeptide having detectable enzyme activity. The kit may also comprise an enzyme substrate for detecting enzyme activity.

[0016] In any embodiment, the kit may further include a housing having at least one wall forming an opening into a compartment, wherein the housing is sized to accommodate the test microorganism and / or enzyme substrate. In some embodiments of the kit described above, the genetically modified test microorganism and / or enzyme substrate may be disposed within the housing. In any embodiment of the kit described above, the genetically modified test microorganism may belong to a genera selected from Bacillus, Geobacillus, Clostridium, and any combination of two or more of the aforementioned genera. In any embodiment of the kit described above, the genetically modified test microorganism may be mounted on a vector. In any embodiment of the kit described above, the kit may further comprise a liquid culture medium suitable for dissolving or suspending the test microorganism and enzyme substrate, the enzyme substrate being reacted with the enzymatic activity of the chimeric protein to form a detectable product.

[0017] Additional details of these and other embodiments are shown in the accompanying drawings and description below. Other features, objectives, and advantages will become apparent from the specification and drawings and from the appended claims.

[0018] In this document, the terms "biosterilization process indicator," "sterilization process bio indicator," "sterilization process indicator," "bio indicator," "BI," and "indicator" are used interchangeably. Similarly, in this document, the terms "complete set bio indicator," "complete set sterilization process indicator," "complete set bio indicator," and "SCBI" are used interchangeably.

[0019] As used herein, the term "biosterilization process indicator" refers to an article of manufacture and / or a device (e.g., a sterilization chamber, autoclave, or autoclave) used to evaluate the effectiveness of a sterilization process. The article of manufacture contains a variety of test microorganisms (e.g., genetically modified test microorganisms as described herein), selected because they possess inherent resistance to the sterilizing agents used in the sterilization process (e.g., dry heat, moist heat, sterilizing agent gases). The test microorganisms are placed on or inside the article of manufacture. Non-limiting examples of articles that can be used as biosterilization process indicators include containers such as tubes, cuvettes, bottles, or micropores; or carrier substrates such as plastic films, paper (e.g., filter paper), glass slides, or metal or ceramic specimens. "Biosterilization process indicator" includes "all-in-one biosterilization process indicator."

[0020] As used herein, a “complete set of biosterilization process indicators” refers to a biosterilization process indicator that includes all components necessary to assess the survival of the genetically modified test microorganism contained therein to determine the lethality of the sterilization process (e.g., shell, test microorganism, liquid culture medium, indicator reagents (e.g., pH indicator, luciferase substrate)).

[0021] The numbers E5, E6, and E7 can be associated with 10 in this article. 5 10 6 and 10 7 Used interchangeably.

[0022] The term “comprising” and its variations (e.g., including, etc.) are not restrictive when these terms appear in the specification and claims.

[0023] As used herein, unless the context clearly indicates otherwise, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably.

[0024] Furthermore, in this document, the numerical range referenced by the endpoints includes all numerical values ​​contained within that range (e.g., 500 to 7000 includes 500, 530, 551, 575, 583, 592, 600, 620, 650, 7000, etc.).

[0025] The terms "preferred" and "ideally" refer to embodiments of the invention that provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of the invention.

[0026] As used herein, the term "openable container" refers to a container that can be actuated when needed to release its contents. A container can be actuated, for example, by fracturing the container, removing or removing a stopper, actuating a valve to change it from a "closed" state to an "open" state, or otherwise damaging at least a portion of the container.

[0027] The term "fragile container" refers to any container that can be acted upon to release its contents, such as by breaking, puncturing, smashing, cutting, etc.

[0028] As used herein, the term "test microorganism" refers to a microorganism used in a method for determining the effectiveness of a sterilization process. Generally, test microorganisms are selected for this method because they exhibit relative resistance to the microbial action of the sterilization process. Spore-form microorganisms are frequently used as test microorganisms because they possess relatively high resistance to the sterilization process. Test microorganisms are typically derived from (e.g., progeny) naturally occurring (i.e., naturally occurring without human modification) microorganisms, including, for example, bacteria.

[0029] As used herein, the term "genetically modified test microbe" refers to a test microbe that has been genetically modified to include a recombinant gene encoding a non-naturally occurring chimeric protein.

[0030] Unless otherwise specified, all scientific and technical terms used herein have their common meaning in the art. The definitions provided herein are intended to aid in understanding certain terms frequently used in this application and are not intended to exclude reasonable interpretations of those terms within the context of this disclosure.

[0031] Unless otherwise specified, all numerical values ​​in the specification and claims expressing characteristic dimensions, quantities, and physical properties should in all cases be understood to be modified by the term "about". Therefore, unless stated to the contrary, the numerical parameters listed in the foregoing specification and appended claims are approximations, which may vary depending on the desired characteristics sought by a person skilled in the art using the teachings disclosed herein. At a minimum, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant digits reported and by applying customary rounding. While the ranges and parameters shown within the broad scope of the invention are approximations, the values ​​shown in specific embodiments are reported as accurately as possible. However, any numerical value inherently contains a certain degree of error, which must be caused by the standard deviation present in their respective experimental measurements.

[0032] As understood in context, the terms “proximity” and “closeness” refer to the relative positions of two elements (such as, for example, two layers) that are close to each other and may or may not need to be in contact with each other or may have one or more layers separating the two elements.

[0033] The phrase “and / or” should be understood to mean “any one or both” of the elements so connected, that is, elements that exist together in some cases and separately in others. In addition to the elements specifically indicated by the “and / or” clause, other elements may optionally exist, whether related to or unrelated to those specifically indicated, unless explicitly stated otherwise. Thus, as a non-limiting example, when used with open-ended language such as “includes,” a reference to “A and / or B” may in one embodiment refer to A without B (optionally including elements other than B); in another embodiment, refer to B without A (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so on.

[0034] When the phrase “at least one” refers to a list of one or more elements, it should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily at least one of every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically specified in the list of elements referred to by the phrase “at least one,” whether related to or unrelated to those specifically specified elements. Thus, as a non-limiting example, in one embodiment, “at least one of A and B” (or in other words, “at least one of A or B”, or in other words, “at least one of A and / or B”) may mean at least one, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, it means at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, it means at least one, optionally including more than one A and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0035] The above description of the invention is not intended to describe every disclosed embodiment or every implementation of the invention. The following description illustrates exemplary embodiments in more detail. Guidance is provided in several places throughout this application by way of a list of embodiments that can be used in various combinations. In each case, the cited list is used only as a representative group and should not be construed as an exclusive list. Attached Figure Description

[0036] Figure 1 This is a schematic gene map of plasmid pJet1.2.

[0037] Figure 2 This is a schematic gene map of plasmid pSTE12.

[0038] Figure 3 This is a partially disassembled cross-sectional view of the complete set of bioindicators disclosed herein.

[0039] Figure 4 yes Figure 2 A cross-sectional view along line 3-3 of the complete set of bio-indicators.

[0040] Figure 5 yes Figures 3 to 5 An exploded perspective view of the complete set of bioindicators. Detailed Implementation

[0041] Before explaining any embodiment of this disclosure in detail, it should be understood that the invention, in its application, is not limited to the construction details and component arrangements mentioned in the following description or shown in the following drawings. The invention can have other embodiments and can be practiced or implemented in various ways. Moreover, it should be understood that the wording and terminology used herein are for illustrative purposes and should not be considered limiting. The use of "comprising," "including," or "having," and variations thereof, herein is intended to cover the items listed thereafter and their equivalents and additional items. It should be understood that other embodiments may be adopted and structural or logical changes may be made without departing from the scope of this disclosure. Furthermore, terms such as "front," "rear," "top," and "bottom" are used only to describe elements related to each other and in no way imply a specific orientation of the device, to indicate or imply a necessary or desired orientation of the device, or to specify how, how, how, how, how, how, how, or how to position the invention described herein in use.

[0042] This disclosure generally relates to sterilization process indicators, kits containing genetically modified test microorganisms used as sterilization process indicators, and methods of using them. The complete set of biological indicators disclosed herein includes all components necessary for assessing the survival of the genetically modified test microorganisms contained therein, and can be used to determine the lethality of sterilization processes using, for example, moist heat (steam), ethylene oxide, hydrogen peroxide, ozone, or combinations thereof as sterilizing agents.

[0043] The genetically modified test microorganisms used in the methods or complete-use bioindicators of this disclosure are genetically modified to include a functional fusion gene encoding a non-naturally occurring chimeric protein. A chimeric protein is a protein translated from a gene generated by linking portions of two or more genes initially encoding separate proteins. Translation of the resulting gene produces a single polypeptide having functional properties derived from each original protein. Therefore, the chimeric protein of this disclosure comprises a first segment and a second segment adjacent to the first segment. The first segment includes a chimeric protein portion adjacent to the N-terminal amino acid of the chimeric protein. The second segment includes a chimeric protein portion adjacent to the C-terminal amino acid of the chimeric protein.

[0044] The first segment of the chimeric protein contains at least a portion of a first polypeptide, which is normally present in spores (e.g., spores of the test microorganism) and can be present in relatively high amounts in the spores. Many genes exist in sporogenic microorganisms whose expression increases during sporulation, leading to increased levels of protein products derived from these genes. These genes include, for example, ysxE, yutH, cotE, cotF, cotH, cotS, cotF, gerE, sspA, sspB, sspD, and sspE. Non-limiting examples of suitable first polypeptides encoded by such genes include small acid-soluble spore proteins, spore coat proteins, and sporulation-associated GTP-binding proteins. Advantageously, using chimeric proteins produced during sporulation ensures the presence of enzymatic activity of the chimeric protein in the spores when they are exposed to the sterilization process, and if the spores survive the sterilization process, there is no delay in detecting enzyme activity (for the biosynthesis of the chimeric protein after the spores survive the sterilization process). A further advantage is that, because the first polypeptide can contain a significant portion of the protein content of the spore, the enzymatic activity of the chimeric protein will also be present in a relatively high amount, thus enabling extremely rapid detection of any spores containing the chimeric protein that survive the sterilization process.

[0045] The portion of the first polypeptide in the chimeric protein must be sufficient to ensure that the chimeric protein is allocated to the spores during sporulation of the genetically modified test microorganism. In some embodiments, the portion of the first polypeptide normally present in the spores comprises the entire first polypeptide. In some embodiments, the portion of the first polypeptide normally present in the spores is smaller than the entire first polypeptide (e.g., representing at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 40%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 2%, or at least 1%).

[0046] The second segment of the chimeric protein contains at least a portion of a second polypeptide normally present in the spore, possessing detectable enzymatic activity. Detectable enzymatic activity is well known in the field of modular bioindicators, including, for example, modular bioindicators with rapid readout capabilities (see, for example, U.S. Patents 5,223,401, 5,252,484, 6,623,955, 6,897,059, and 6,566,090; each of which is incorporated herein by reference in its entirety). Many genes in microorganisms are translated into polypeptides possessing detectable enzymatic activity. These genes include, for example, mall, bglH, lacZ, phoA, phoD, and pepA. Non-limiting examples of suitable second polypeptides encoded by such genes include α-glucosidase, α-galactosidase, lipase, esterase, acid phosphatase, alkaline phosphatase, protease, aminopeptidase, chymotrypsin, β-glucosidase, β-galactosidase, α-glucuronidase, β-glucuronidase, phosphorylhydrolase, α-mannosidase, β-mannosidase, αL-fucosidase, leucine aminopeptidase, αL-arabinofuranyl, cysteine ​​aminopeptidase, valine aminopeptidase, β-xylosidase, α-L-iduronidase, glucanase, cellobiosidase, cellulase, α-arabinosidase, polysaccharidease, sulfatase, butyrate esterase, glycosidase, and arabinosidase.

[0047] This portion of the second polypeptide in the chimeric protein must be sufficient to ensure that the chimeric protein retains the enzymatic activity of the second polypeptide.

[0048] In some implementations, the fusion gene also includes genetic information of a linker polypeptide that connects the first polypeptide to the second polypeptide. The linker polypeptide separates the first polypeptide from the second polypeptide, thus helping the first and second polypeptides to fold correctly within the spore, thereby preserving their respective functional activities.

[0049] A fusion gene is introduced into a test microorganism to produce a genetically modified test microorganism. Methods for introducing genes into microorganisms are well known in the art. For example, the fusion gene can be integrated into a plasmid vector, which can be transferred into the test microorganism by a method called transformation. From there, the fusion gene can be retained as part of an extrachromosomal replicon (e.g., the plasmid used as a vector), or the fusion gene can be integrated into the chromosomal replicon of the test microorganism via in vivo recombination. In some embodiments, the integration of the fusion gene into the genome can be mediated by insertion sequences known in the art. In some embodiments, the plasmid vector can be constructed such that the flanks of the fusion gene are DNA sequences from the test microorganism, and the fusion gene is integrated into the genome via homologous recombination.

[0050] Plasmids are used to transfer genes into sporulating microorganisms. Non-limiting examples of plasmids that can be used to introduce fusion genes into sporulating test microorganisms include pJET1.2, pNW33N, pJF751, and the Geobacillus shuttle plasmid described by Reeve et al. (“The Geobacillus Plasmid Set: A Modular Toolkit for Thermophile Engineering”, ACS Synthetic Biology, 2016, Vol. 5, pp. 1342-1347, the full text of which is incorporated herein by reference). Cloning vectors and techniques for transferring genes into sporulating microorganisms are known in the art and described, for example, in “Genetic engineering of Geobacillus spp.” (…). The full texts of the two articles, “High osmolarity improves the electro-transformation efficiency of gram-positive bacteria Bacillus subtilis and Bacillus licheniformis” (Xue et al., Journal of Microbiological Methods, 1999, Vol. 111, pp. 31-39), are incorporated herein by reference.

[0051] Plasmids used to construct the engineered microorganisms of this disclosure may contain one or more DNA sequences serving as origins of replication (ori). Plasmids may contain one or more genetic markers. Plasmids may contain multiple adapters or multiple cloning sites (MCS), which may be relatively short regions containing one or more restriction sites that allow insertion of DNA fragments. Plasmids may contain one or more genes that provide selection markers to induce the test organism to retain the plasmid. Selection markers may contain antibiotic resistance genes and / or genes with nutritional capabilities. Plasmids may include conjugative plasmids containing tra-genes that perform the conjugation process, i.e., the sexual transfer of the plasmid to another bacterium. Transformation of the test microorganisms with recombinant plasmids containing genes capable of expressing fusion proteins can be performed using techniques known in the art, such as those described, for example, by Reeve et al. Maintaining the selective pressure of the plasmid in the transformed test microorganisms can be achieved using techniques known in the art (e.g., antibiotic selection).

[0052] Switch to the attached image. Figure 1 A gene map of one embodiment of the plasmid (pJET1.2) is shown, which is used as a vector to introduce genes (e.g., genes encoding fusion proteins) into test microorganisms (e.g., *Bacillus stearothermophilus*). The plasmid includes a β-lactamase gene (AmpR), a chloramphenicol acetyltransferase gene (CAT), an origin of replication (ori), and several unique restriction sites (not shown) that can be used for cloning purposes. The unique PstI and BbsI restriction sites described in Example 1 are shown in... Figure 1 middle.

[0053] Insert sequences have been used to transfer genes into the genomes of sporulating microorganisms. Non-limiting examples of insert sequences that can be used to introduce fusion genes into sporulating test microorganisms include, for example, members of the IS4 and IS21 insert sequence families as described in U.S. Patent No. 9,416,393, the full text of which is incorporated herein by reference.

[0054] In some embodiments, the fusion gene includes regulatory elements that promote the expression of the chimeric protein in a genetically modified host microorganism. These regulatory elements include, but are not limited to, at least one of a transcription promoter, a ribosome binding site, a translation start codon, a signal sequence, and a translation stop codon. In some embodiments, one or more of the regulatory elements are regulatory elements present in a natural gene that expresses a first polypeptide normally present in the spore.

[0055] As indicated above, a complete set of biosterilization process indicators is now available for evaluating the effectiveness of various sterilization processes, including, for example, steam sterilization processes using temperatures of 121°C, 132°C, 134°C, or 135°C; ethylene oxide sterilization processes; peroxide sterilization processes; and ozone sterilization processes.

[0056] Other components used in the complete set of bioindicators disclosed herein are discussed in more detail below.

[0057] case

[0058] For non-limiting examples of housings suitable for the complete set of bioindicators applicable to this disclosure, see U.S. Patent Nos. 3,661,717, 5,223,401, and 6,623,955; and U.S. Patent Application Publication Nos. 2013 / 0302849 and 2014 / 0349335; each of these patents is incorporated herein by reference in its entirety. Generally, a housing refers to a container, typically an outer container, that houses other components of a bioindicator and has walls impermeable to sterilizing agents. The housing may be located within a process challenge device or may be the process challenge device itself. In some embodiments, the housing may have dimensions suitable for producing a flat or generally planar bioindicator. This disclosure covers housings of any shape and size.

[0059] The housing includes at least one opening (sterilizer path) that allows a sterilizing agent to flow into the interior of the housing. In some embodiments, the housing may include a body with the opening and a cap that closes the opening. In some embodiments, the cap may be able to completely seal the housing and eliminate any fluid communication between the interior of the housing and the surrounding environment (e.g., closing the sterilizer path). Generally, the cap has an open position in which an opening (e.g., a gap) exists between the cap and the body of the container, allowing liquids or gases (e.g., sterilizing agents) to flow into and out of the interior of the housing. The cap also has a closed position in which the opening is sealed and any fluid flow through the gap is eliminated. In other embodiments, the cap may include vents that allow sterilizing agents to pass into the interior of the housing and form additional sterilizer paths, even when the cap is present and in the closed position. However, in other preferred embodiments, when the cap includes vents, placing the cap in the closed position simultaneously closes: (a) the gap between the cap and the body of the container and (b) the vents present on the cap, thereby substantially closing the sterilizer path.

[0060] In other embodiments, the cover may lack a vent, and when the cover is in the open position, the only sterilizing agent path may pass through the space between the cover and the body of the housing (or through another opening or vent (if present on the body)). In some embodiments, if vents exist on the housing, they are located on the cover. In embodiments where there are no other openings besides the opening between the cover and the body of the housing, placing the cover in the closed position completely seals the interior of the housing, thereby preventing fluid communication between the interior of the housing and the surrounding environment. In those embodiments, the sterilizing agent path may be sealed when the cover is in the closed position.

[0061] Genetically modified test microorganisms

[0062] The genetically modified test microorganisms used in the methods or kit-mounted bioindicators of this disclosure are sporulating microorganisms that are particularly resistant to a given sterilization process. In some embodiments, the methods and kit-mounted bioindicators of this disclosure include live cultures of known species of microorganisms, typically in the form of microbial spores. Spores (e.g., bacterial spores) are used at least in part rather than the vegetative form of the microorganism because vegetative microorganisms are known to be relatively easily killed by the sterilization process.

[0063] The methods and modular biological indicators disclosed herein include multiple genetically modified test microorganisms disposed therein (e.g., within a housing). The genetically modified test microorganisms may belong to one or more species. Typically, the biological indicator contains a predetermined amount of at least 10 3 At least 10 4 At least 10 5 At least 10 6 At least 10 7 Or at least 10 8 A genetically modified test microorganism.

[0064] By way of example only, this disclosure describes the microorganisms used in the methods and kit-mounted bioindicators as “spores”; however, it should be understood that the type of microorganism (e.g., spores) used in a particular embodiment of the bioindicator is selected for its tolerance to the contemplated specific sterilization process (greater tolerance than microorganisms normally present on the article to be sterilized, so that the inactivation of the genetically modified test microorganism indicates successful sterilization). Therefore, the genetically modified test microorganisms included in embodiments of the methods or kit-mounted bioindicators of this disclosure used with a specific sterilization process (or sterilizing agent) may differ from those in embodiments of the methods or bioindicators of this disclosure used with different sterilization processes (or sterilizing agents) (e.g., different species).

[0065] In some embodiments, the genetically modified test microorganism may include, but is not limited to, at least one of the following: Geobacillus stearothermophilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus atrophaeus, Bacillus megaterium, Bacillus scoagulans, Clostridium sporogenes, Bacillus pumilus, or a combination thereof.

[0066] enzyme substrate

[0067] The complete bioindicator disclosed herein includes an enzyme substrate for detecting enzyme activity. The enzyme activity of the chimeric protein should be such that it retains sufficient activity to react with a suitable enzyme substrate for one hour or less after a sublethal sterilization cycle of the genetically modified test microorganism, but is inactivated or significantly reduced after a lethal sterilization cycle of the genetically modified test microorganism.

[0068] In the context of this application, an enzyme substrate includes a substance or mixture of substances that, when acted upon by an enzyme, converts into an enzyme-modified product. While preferred substrates produce compounds capable of fluorescent detection, in other embodiments, the product of enzymatic reaction may be a luminescent or colored material. However, in other embodiments, the enzyme substrate may consist of a compound that, upon reaction with an enzyme, produces a product that reacts with additional compounds or compositions to produce a luminescent, fluorescent, or colored material. Preferably, the substrate should not spontaneously decompose or convert into a detectable product for detecting enzyme activity during the sterilization process or during subsequent incubation. For example, in apparatus used for monitoring steam and dry heat sterilization, the substrate must be stable at temperatures between about 20°C and 180°C.

[0069] Generally, there are two basic types of enzyme substrates that can be used in the methods and kitted biological indicators of this disclosure. The first type of substrate may be fluorescent (or chromogenic) and may be given by a chemical formula such as AB. When acted upon by an enzyme, AB decomposes into products A and B. B may be, for example, fluorescent or chromogenic. A specific example of this type of fluorescent substrate is a salt of 4-methylumbelliferyl ketone. Other fluorescent substrates of this type include derivatives of 4-methylumbelliferyl ketone, 7-amido-4-methylcoumarin (7-AMC), indophenol, and fluorescein. An example of this type of chromogenic substrate is 5-bromo-4-chloro-3-indolyl phosphate. In the presence of a phosphatase, the substrate will decompose into indigo and phosphate. Other chromogenic substrates of this type include derivatives of 5-bromo-4-chloro-3-indolyl, nitrophenol, and phenolphthalein.

[0070] The second type of substrate can be given, for example, by the chemical formula CD, which will be converted into C and D by a specific enzyme. However, in this case, neither C nor D will fluoresce or be colored, but either C or D can further react with compound Z to produce a fluorescent or colored compound, thus indicating enzyme activity. A specific fluorescent example of this type is the amino acid lysine. In the presence of lysine decarboxylase, lysine loses a CO2 molecule. The remaining portion of lysine is then called cadaverine, which is strongly basic. A basic indicator, such as 4-methylumbelliferone, can be incorporated, and this basic indicator will fluoresce in the presence of a strong base. A chromogenic substrate of this type would be 2-naphthyl phosphate. Phosphatase reacts with this substrate to produce β-naphthol. The released β-naphthol reacts with a chromogenic reagent containing 1-diazo-4-benzoylamino-2,5-diethoxybenzene (commercially available as "Glass Blue BB salt" from Sigma Chemical) to produce a violet color.

[0071] As described above, the preferred enzyme substrate in some embodiments is a fluorescent substrate, which is defined herein as a compound capable of being enzymatically modified (e.g., by hydrolysis or other enzymatic actions) to give a derivative fluorophore having a significantly altered or enhanced fluorescence.

[0072] Those skilled in the art will understand that suitable fluorescent compounds are themselves non-fluorescent or meta-fluorescent (i.e., fluorescing in a manner distinctly different from the corresponding enzyme-modified product (e.g., in color or intensity)). In this regard, using appropriate excitation and detection wavelengths in a manner known to users of fluorescence technology will separate the fluorescent signal formed by enzyme modification from any other fluorescence that may exist.

[0073] Non-limiting examples of suitable enzyme substrates may include, for example, coumarin derivatives, including 7-hydroxycoumarin (also known as umbelliferone or 7-hydroxy-2H-benzopyran-2-one) derivatives and 4-methylumbelliferone (7-hydroxy-4-methylcoumarin) derivatives, including: 4-methylumbelliferone-α-D-glucopyranoside, 4-methylumbelliferone-α-D-galactopyranoside, 4-methylumbelliferone heptanoate, 4-methylumbelliferone palmitate, 4-methylumbelliferone oleate, 4-methylumbelliferone acetate, 4-methylumbelliferone nonanoate, 4-methylumbelliferone octanoate, 4-methylumbelliferone butyrate, 4-methylumbelliferone-β-carboxylic acid. -D-Cellulobiose, 4-methylumbelliferone acetate, 4-methylumbelliferone phosphate, 4-methylumbelliferone sulfate, 4-methylumbelliferone-β-trimethylammonium chloride cinnamic acid, 4-methylumbelliferone-β-DN,N',N"-triacetylchitosan, 4-methylumbelliferone-β-D-xyloside, 4-methylumbelliferone-N-acetyl-β-D-aminoglucosinolate, 4-methylumbelliferone-N-acetyl-α-D-aminoglucosinolate, 4-methylumbelliferone propionate, 4-methylumbelliferone stearate, 4-methylumbelliferone-α-L-arabinofuranoside, 4-methylumbelliferone-α-L-arabinoside;Methylumbelliferyl-β-DN,N'-diacetylchitoside, 4-methylumbelliferyl transoleate, 4-methylumbelliferyl-α-D-mannopyranoside, 4-methylumbelliferyl-β-D-mannopyranoside, 4-methylumbelliferyl-β-D-fucoside, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-β-L-fucoside, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-β-D-galactoside, 4-trifluoromethylumbelliferyl-β-D-galactose Glycosides, 4-methylumbelliferone-α-D-glucosinolates, 4-methylumbelliferone-β-D-glucosinolates, 4-methylumbelliferone-7,6-sulfono-2-acetamido-2-deoxy-β-D-glucosinolates, 4-methylumbelliferone-β-D-glucuronic acid, 6,8-difluoro-4-methylumbelliferone-β-D-glucuronic acid, 6,8-difluoro-4-methylumbelliferone-β-D-galactoside, 6,8-difluoro-4-methylumbelliferone phosphate, 6,8-difluoro-4-methylumbelliferone-β-D-xylobiose. The second substrate can also be a derivative of 7-amido-4-methylcoumarin, including: Ala-Ala-Phe-7-amido-4-methylcoumarin, Boc-Gln-Ala-Arg-7-amido-4-methylcoumarin hydrochloride, Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin, Boc-Val-Pro-Arg-7-amido-4-methylcoumarin hydrochloride, D-Ala-Leu-Lys-7-amido-4-methylcoumarin, L-alanine-7-amido-4-methylcoumarin trifluoroacetate, L-methionine-7-amido-4-methylcoumarin trifluoroacetate, L-tyrosine-7-amido-4-methylcoumarin, Lys-Ala-7-amido-4-methylcoumarin dihydrochloride, N-p-toluenesulfonyl-Gly- Pro-Arg-7-amido-4-methylcoumarin hydrochloride, N-succino-Ala-Ala-Phe-7-amido-4-methylcoumarin, N-succino-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin, N-succino-Ala-Phe-Lys-7-amido-4-methylcoumarin acetate, N-succino-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin, D-Val-Leu-Lys-7-amido-4-methylcoumarin, Fmoc-L-glutamic acid 1-(7-amido-4-methylcoumarin), Gly-Pro-7-amido-4-methylcoumarin hydrobromide, L-leucine-7-amido-4-methylcoumarin hydrochloride, L-proline-7-amido-4-methylcoumarin hydrobromide;Other 7-hydroxycoumarin derivatives include 3-cyano-7-hydroxycoumarin (3-cyanoumbelliferone) and 7-hydroxycoumarin-3-carboxylic acid esters, such as ethyl 7-hydroxycoumarin-3-carboxylic acid, methyl 7-hydroxycoumarin-3-carboxylic acid, 3-cyano-4-methylumbelliferone, and 3-(4-imidazolyl)umbelliferone; fluorescein derivatives include: 2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein, 2',7'-bis-(2-carboxypropyl)-5-(and-6-)-carboxyfluorescein, 5-(and-6)-carboxynaphthalenefluorescein, lithofluorescein, 2',7'-dichlorofluorescein diacetate, 5(6)-carboxyfluorescein, 5(bromomethyl)fluorescein, 5-(iodoacetamido)fluorescein, 5-([4,6-dichlorotriazine-2-yl]amino)fluorescein hydrochloride, 6-carboxyfluorescein, eosin Y, diacetic acid fluorescein 5-maleimide, fluorescein-O'-acetic acid, O'-(carboxymethyl)fluorescein amide, anthracene fluorescein, rhodols, halogenated fluorescein; derivatives of rhodamine, including: tetramethylrhodamine, carboxytetramethylrhodamine, carboxy-X-rhodamine, sulfonylrhodamine 101 and rhodamine B; fluoroglucosamine derivatives; derivatives of benzanthracene dyes, including: seminaphthorhodafluorescein, carboxyseminaphthorhodafluorescein, seminaphthorhodafluorescein, seminaphthorhodafluorescein; derivatives of cyanine, including sulfonated pentamethinecyanine and septamethinecyanine.

[0074] The concentration of the enzyme substrate present in the method or a complete biological indicator (e.g., when dissolved and / or suspended in an aqueous liquid culture medium within the biological indicator) depends on the specific enzyme substrate and enzyme identity, the amount of enzyme product that must be generated for visual or instrumental detection, and the amount of time willing to be waited to determine the presence of an active enzyme in the reaction mixture. Preferably, the amount of enzyme substrate is sufficient to react with any residual active enzyme present for a period of approximately eight hours after sterilization cycles, resulting in the production of at least 10 -8 Mohr's enzyme-modified product. In the case of an enzyme substrate that is a 4-methylumbelliferyl ketone derivative, the inventors have found that its concentration in the aqueous liquid culture medium disclosed herein is preferably about 10. -5 With 10 -3Between moles. In some embodiments, 4-methylumbelliferyl ketone-α-D-glucoside may be used in aqueous mixtures, for example, at concentrations of about 0.05 g / L to about 0.5 g / L (such as about 0.05 g / L, about 0.06 g / L, about 0.07 g / L, about 0.08 g / L, about 0.09 g / L, about 0.1 g / L, about 0.15 g / L, about 0.2 g / L, about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, about 0.4 g / L, about 0.45 g / L, about 0.5 g / L).

[0075] pH indicator dye

[0076] In any embodiment, the all-in-one biological indicator of this disclosure may optionally include a pH indicator dye disposed within a housing (e.g., disposed within a compartment). In some embodiments, as described in U.S. Provisional Patent Application No. 62 / 990,483, filed March 17, 2020, entitled “Immobilized pH Indicator for Biological Indicator Growth Indication,” the entire contents of which are incorporated herein by reference. In any embodiment, the indicator dye may be a pH indicator suitable for detecting biological activity (e.g., fermentation of carbohydrate nutrients). The indicator dye may be selected according to criteria known in the art, such as pH range, compatibility with biological activity, and solubility. In some embodiments, a salt form of the pH indicator may be used, for example, to increase the solubility of the pH indicator in aqueous mixtures. Non-limiting examples of suitable pH indicator dyes include, for example, thymol blue, golden orange OO, methyl yellow, methyl orange, bromophenol blue, bromocresol green, methyl red, bromothymol blue, phenol red, chlorophenol red, neutral red, naphtholphthalein, phenolphthalein, thymolphthalein, alizarin yellow, golden orange O, nitroamine, trinitrobenzoic acid, thymol blue, bromophenol blue, tetrabromophenol blue, bromocresol green, bromocresol violet, methyl red, bromothymol blue, Congo red, and cresol red. In some embodiments, the pH indicator dye is anionic in solutions with a pH of approximately neutral.

[0077] In some embodiments, the pH indicator dye produces a color change as the pH decreases, thereby indicating the growth of the genetically modified test microorganism. In some embodiments, the pH indicator dye is bromocresol purple. pH indicators can be used to detect biological activities, such as the fermentation of nutrients (e.g., carbohydrates set in a bioindicator) into an acidic end product (indicating the survival of the genetically modified test microorganism). For example, these activities can indicate the presence or absence of live spores after the bioindicator has undergone a sterilization process. For example, bromocresol purple can be used in aqueous mixtures at a concentration of about 0.03 g / L.

[0078] The combination of bromocresol purple and 4-methylumbelliferyl-α-D-glucoside represents a preferred combination of enzyme substrate and pH indicator dye in the articles or methods according to this disclosure, but other combinations are contemplated within the scope of this disclosure.

[0079] Complete set of biological indicators

[0080] The genetically modified test microorganisms described herein can be used in a variety of bioindicators known in the art to produce bioindicators or complete sets of bioindicators according to this disclosure. The resulting bioindicators or complete sets of bioindicators are particularly useful for evaluating the effectiveness of sterilization processes (e.g., steam sterilization processes).

[0081] For example, the complete-use bioindicator of U.S. Patent No. 3,661,717, which is incorporated herein by reference in its entirety, can be modified to use the genetically modified test microorganisms described herein. In some embodiments, the genetically modified test microorganisms may be provided on a carrier substrate or on the inner surface of the bioindicator housing.

[0082] Additionally, the complete-use bioindicators of U.S. Patent Nos. 5,223,401 and 6,623,955, which are incorporated herein by reference in their entirety, can be modified to use the genetically modified test microorganisms described herein. In some embodiments, the genetically modified test microorganisms may be provided on a carrier substrate or on the inner surface of the bioindicator housing.

[0083] Furthermore, the all-in-one bioindicator of U.S. Patent Application Publication No. US 2013 / 0302849, which is incorporated herein by reference in its entirety, can be modified to use the genetically modified test microorganisms described herein. In some embodiments, the genetically modified test microorganisms may be provided on a carrier substrate or on the inner surface of the bioindicator housing.

[0084] Those skilled in the art will recognize how to modify other existing biological indicators using the genetically modified test microorganisms of this disclosure to obtain the articles and methods of this disclosure.

[0085] In this disclosure, the process of bringing together spores and culture medium is referred to as “activation” of the modular bioindicator. That is, the term “activation” and its variations, when used in relation to a modular bioindicator, generally refer to fluid communication between one or more genetically modified test microorganisms (e.g., spores) and a liquid culture medium (e.g., an aqueous liquid culture medium containing nutrients and / or enzyme substrates). For example, the modular bioindicator can be described as “activated” when an openable container within the modular bioindicator containing the liquid culture medium is at least partially opened (e.g., broken, perforated, punctured, crushed, fractured, ruptured, etc.), such that the culture medium has been placed in fluid communication with the genetically modified test microorganism.

[0086] Turn to the attached diagram. Figures 3 to 5 Various views of one embodiment of the complete set of bio-indicators 100 according to this disclosure are shown.

[0087] The complete biometric indicator 100 is shown with a housing 10, which includes a compartment 11 and a cover 28. The compartment 11 has at least one wall 12 forming an opening 14. For example, at least one wall may be made of a moisture-impermeable, non-absorbent material (such as glass or plastic). In some preferred embodiments, the compartment is formed of an optically transparent or translucent material.

[0088] The complete bioindicator 100 includes a plurality of genetically modified test microorganisms (e.g., bacterial spores) 17 disposed within a housing 10. For example, the genetically modified test microorganisms 17 may optionally be disposed on a carrier 16 (e.g., a sheet material, such as filter paper strips or polymer membranes), for example as a substantially anhydrous coating. In some embodiments, the carrier 16 is made of a water-impermeable material.

[0089] The complete biological indicator 100 includes a liquid culture medium 20 (e.g., an aqueous liquid culture medium) disposed in an openable container 18. The contents of the openable container 18 (e.g., the liquid culture medium 20) are selectively in communication with a compartment 11 of the housing 10. Figures 3 to 5 In the illustrated embodiment, the openable container 18 is a normally sealed, pressure-openable container, such as a fragile glass ampoule. Those skilled in the art will recognize other suitable openable containers 18 (including some embodiments in which the container is disposed outside the housing 10) and opening devices (e.g., valves, protruding seals; not shown) that provide selective fluid communication between the openable container and the compartment.

[0090] enzyme substrate

[0091] The complete bioindicator 100 includes an enzyme substrate capable of reacting with the activity of a detectable enzyme to form a detectable product. The enzyme substrate ( Figures 3 to 5 (Not shown) is disposed in a container or housing. In some embodiments, the enzyme substrate is dissolved and / or suspended in a liquid culture medium 20 disposed in an openable container 18 disposed in a housing 10. In some alternative embodiments (not shown), the enzyme substrate may be disposed in a dry form (e.g., a dry coating, powder, tablet) in the housing, which may dissolve and / or suspend in the liquid culture medium upon actuation of the openable container. Additionally, upon actuation of the openable container, the liquid culture medium and enzyme substrate may come into contact with the genetically modified test microorganism, thereby facilitating the detection of any detectable products formed by live genetically modified test microorganisms (if present) in the kit bioindicator after exposure to the sterilization process.

[0092] exist Figures 3 to 5 In the illustrated embodiment, the container 18 is held tightly within the compartment, leaving minimal volume of the compartment 10 unoccupied. The container 18 is separated from the wall 12 of the compartment 10 by a carrier 16, thereby providing cavities 24 and 26 between the wall 12 and the container 18. The open end 14 of the compartment 10 is provided with a breathable, bacteria-impermeable sealing member, shown as a sheet 22. The sheet 22 can be sealed, for example, by heat or adhesive, or by means of a cap 28 (in... Figure 4 The cover 28 is shown as being removed from the opening 14 of the compartment 10, and has an orifice 29 passing through it at an adjacent sheet 22. The orifice 29 allows sterilizing agent to enter the housing from the outside. The cover 28 can be formed from various materials (e.g., metals, plastics) using methods well known in the art. During steam sterilization, for example, steam permeates the sheet 22 and through cavities 24 and 26 to contact the genetically modified test microorganism 17 disposed on the carrier 16.

[0093] like Figure 5 As shown, the complete set of bioindicators 100 can be easily assembled by sequentially inserting the carrier 16 (on which the genetically modified test microorganism 17 is disposed) and the container 18 into the opening 14 of the compartment 10 and sealing the opening 14 of the tube with the sheet 22. Before assembly, the genetically modified test microorganism 17 can be deposited onto the carrier 16 as a liquid suspension, and then dried before placing the carrier 16 into the container 10.

[0094] sterilization process

[0095] In at least some sterilization processes where steam is used as the sterilizing agent, high temperatures, such as 121°C, 132°C, 134°C, 135°C, etc., are involved or may be encountered. Additionally, high pressure and / or vacuum, such as 15 psi (1 × 10⁻⁶), may be encountered at different stages within a single given sterilization cycle or in different sterilization cycles. 5 Pa).

[0096] When steam is used as the sterilizing agent, sterilization temperatures may include 121°C, 132°C, 134°C, and 135°C. The complete-use bioindicator is suitable for steam sterilization cycles at each of the aforementioned temperatures, and for each temperature, the cycle may have a different air removal process selected from gravity, pre-vacuum (“pre-vac”), and steam jet pressure pulse (SFPP). Each of these cycles may have a different contact time, depending on the type of instrument / device to be sterilized. In this disclosure, pre-vacuum and SFPP are also referred to as dynamic air removal (DAR) cycles.

[0097] Generally, a sterilization process may include placing the modular bioindicator of this disclosure in a sterilizer (e.g., the sterilization chamber of an automated sterilizer). In some embodiments, the sterilizer includes a sterilization chamber sized to accommodate multiple articles to be sterilized and may be equipped with components for venting air and / or other gases from the chamber and for adding steam to the chamber. The modular bioindicator may be positioned in the most difficult-to-sterilize area of ​​the sterilizer. Alternatively, the modular bioindicator may be positioned in a process challenge device to simulate sterilization conditions where steam may not be delivered directly as in more favorable sterilization scenarios.

[0098] Steam sterilizers can be added to the sterilization chamber after at least a portion of any air or other gases present in the chamber has been purged. Alternatively, steam can be added to the chamber without purging. A series of purging steps can be used to ensure that the steam sterilizer reaches all desired areas within the chamber and contacts all desired products to be sterilized, including integrated biological indicators.

[0099] The complete set of biological indicators can determine the effectiveness of one or more steam sterilization cycles selected from a power set of the following eleven cycles: 121°C gravity, 121°C pre-vacuum, 121°C SFPP, 132°C gravity, 132°C pre-vacuum, 132°C SFPP, 134°C pre-vacuum, 134°C SFPP, 135°C gravity, 135°C pre-vacuum, and 135°C SFPP, preferably within one hour after the complete set of biological indicators is removed from the sterilizer.

[0100] liquid culture medium

[0101] The complete, modular biological indicator disclosed herein includes a liquid culture medium disposed in an openable container, which is disposed within or attached to a housing. Suitable openable containers include, for example, the glass ampoule 18 described in U.S. Patent No. 3,661,717; the inner container 48 described in U.S. Patent No. 5,223,401; the inner compartment 18 and inner containers 48 and 78 described in U.S. Patent No. 6,623,955; and the fragile container 120 described in U.S. Patent Application Publication No. 2013 / 0302849.

[0102] The liquid culture medium may contain one or more of the enzyme substrates mentioned herein, provided that at least one of the enzyme substrates can be converted into a detectable product by the enzymatic activity of a second polypeptide of the fusion protein. In some embodiments, the enzyme substrate is 4-methylumbelliferyl ketone-α-D-glucoside (MUG). In some embodiments, the liquid culture medium may optionally also include a nutrient composition that promotes the germination and / or growth of the genetically modified test microorganism. In some embodiments, the liquid culture medium contains water.

[0103] Optionally, suitable nutrients may be initially provided in the shell in a dry form (e.g., powder, tablet, capsule, film or coating, retained in beads or other carriers, another suitable shape or configuration, or a combination thereof). When combined with a liquid culture medium (e.g., when the kit bioindicator is actuated), the nutrients can contact the genetically modified test microorganisms and promote the growth of any live genetically modified test microorganisms retained in the shell after the kit bioindicator has been exposed to a sterilization process.

[0104] Nutrients may include one or more sugars, including but not limited to glucose, fructose, dextrose, maltose, trehalose, cellobiose, or combinations thereof. Alternatively, nutrients may include complex culture media, such as peptone, tryptone, plant peptone, yeast extract, soybean casein digest, other extracts, hydrolysates, or combinations thereof. In other embodiments, nutrients comprise a combination of one or more complex culture media components and other specific nutrients. Nutrients may also include salts, including but not limited to sodium chloride, potassium chloride, calcium chloride, or combinations thereof. In some embodiments, nutrients may also include at least one amino acid, including but not limited to at least one of methionine, phenylalanine, alanine, tyrosine, and tryptophan.

[0105] As part of the modular bioindicator, a liquid culture medium, optionally containing nutrients, enzyme substrates, and other components, is typically present throughout the sterilization process but remains isolated in an openable container and inaccessible to the genetically modified test microorganism until the modular bioindicator is activated. After the sterilization process is completed and the effectiveness of sterilization is determined using the modular bioindicator, the liquid culture medium is placed in contact with the genetically modified test microorganism and nutrients to create a mixture. In this disclosure, placing the liquid culture medium in contact with the genetically modified test microorganism includes activating the openable container, causing the liquid culture medium to be released and contacted with the genetically modified test microorganism. The process may include mixing the liquid culture medium with the genetically modified test microorganism, such as by manually or mechanically agitating the housing of the modular bioindicator to ensure thorough mixing of the liquid culture with the genetically modified test microorganism.

[0106] In this disclosure, the process of bringing together a genetically modified test microorganism and a liquid culture medium is referred to as “activation” of the kit bioindicator. That is, the term “activation” and its variations, when used in relation to a kit bioindicator, generally refer to contacting one or more genetically modified test microorganisms (e.g., spores) with a liquid culture medium (optionally containing an enzyme substrate). For example, a kit bioindicator can be described as “activated” when an openable container within a kit bioindicator containing liquid culture medium is at least partially broken, perforated, punctured, crushed, fractured, or ruptured, such that the culture medium has been placed in fluid communication with the genetically modified test microorganism. In other words, a kit bioindicator is activated when the genetically modified test microorganism has been contacted with a liquid culture medium previously contained separately from the genetically modified test microorganism.

[0107] In some preferred embodiments, the mixture resulting from the mixing of liquid culture medium and the genetically modified test microorganisms after activation remains separated within the housing of the modular bioindicator after sterilization cycles have been completed, and no additional reagents or components are added to it during or after activation. If at least one of the genetically modified test microorganisms is viable, the enzymatic activity of the chimeric protein can react with the enzyme substrate, which can produce a detectable compound (e.g., a fluorescently detectable compound).

[0108] In some embodiments, the liquid culture medium may include a buffer solution, such as the buffer solution described in U.S. Patent Application No. 62 / 964,369, filed January 22, 2020, entitled “Self-Contained Biological Indicator with Salt Compound,” the entire contents of which are incorporated herein by reference. The ionic conditions of the buffer solution should be such that the enzyme and enzyme substrate (if present in the self-contained biological indicator) are unaffected. In some embodiments, the buffer solution is used as part of the liquid culture medium, such as a phosphate buffer (e.g., phosphate-buffered saline solution, potassium phosphate, or dipotassium hydrogen phosphate), tris(hydroxymethyl)aminomethane-HCl solution, or acetate buffer, or any other buffer known in the art suitable for sterilization. If a luciferase substrate and / or chromogenic enzyme substrate is used as part of the self-contained biological indicator, then the buffer solution suitable for the self-contained biological indicator of the present invention should be compatible with the enzyme substrate.

[0109] If an enzyme substrate is present in the liquid culture medium before or after activation of the modular bioindicator, the concentration of the enzyme substrate depends on the characteristics of the specific substrate and the enzymatic activity of the chimeric protein; the amount of enzyme product that must be produced, which can be detected visually or by instruments; and the amount of time one is willing to wait to determine the presence of an active enzyme in the reaction mixture. Preferably, the amount of enzyme substrate is sufficient to react with any residual active enzyme present in the chimeric protein within less than three hours or less than one hour after the sterilization cycle, resulting in a local concentration of at least 10. -8 The enzyme-modified product in moles. In the case of a 4-methylumbelliferyl ketone derivative as the enzyme substrate, the inventors have found that its concentration in an aqueous buffer solution is preferably about 10. -5 With 10 -3 Between moles.

[0110] In some embodiments, the complete bioindicator may include an additional indicator compound (in addition to an enzyme substrate that produces a compound capable of fluorescent detection) that facilitates the detection of another metabolic activity of a genetically modified test microorganism (e.g., spores). This additional metabolic activity may also be enzymatic activity. Non-limiting examples of indicator compounds include chromogenic enzyme substrates (e.g., observable in the visible spectrum), pH indicators, redox indicators, chemiluminescent enzyme substrates, dyes, and combinations of any two or more of the aforementioned indicator compounds.

[0111] In some embodiments, the additional indicator is a pH indicator that produces a color change as the pH decreases, thereby indicating the growth of the genetically modified test microorganism. In some embodiments, the pH indicator is bromocresol purple. The pH indicator can be used to detect secondary biological activities such as the fermentation of carbohydrates into acidic end products (indicating the survival of the genetically modified test microorganism) and enzymatic biological activities such as α-D-glucosidase activity. For example, these activities can indicate the presence or absence of live genetically modified test microorganisms after contact sterilization with the integrated bioindicator. For example, bromocresol purple can be used in aqueous mixtures at a concentration of about 0.03 g / L. 4-Methylumbelliferone-α-D-glucoside can be used in aqueous mixtures, for example, at concentrations of about 0.05 to about 0.5 g / L (such as about 0.05 g / L, about 0.06 g / L, about 0.07 g / L, about 0.08 g / L, about 0.09 g / L, about 0.1 g / L, about 0.15 g / L, about 0.2 g / L, about 0.25 g / L, about 0.3 g / L, about 0.35 g / L, about 0.4 g / L, about 0.45 g / L, about 0.5 g / L).

[0112] Methods for evaluating the effectiveness of sterilization processes

[0113] In another aspect, this disclosure provides a method for determining the effectiveness of a sterilization process. A bioindicator, including the complete bioindicator of this disclosure (e.g., an SCBI containing a genetically modified test microorganism of *Bacillus stearothermophilus* strain), can be used in said method to monitor the effectiveness of one or more types of sterilization procedures, including sterilization procedures that, for example, use steam (e.g., pressurized steam) as a sterilizing agent. The term "sterilization process bioindicator" as used with reference to the method of this disclosure is broadly used for articles containing or comprising a plurality of genetically modified test microorganisms and intended to verify the effectiveness of a sterilization process. A sterilization process bioindicator includes, for example, a container holding a substrate on which the genetically modified test microorganism of this disclosure is immobilized (e.g., a strip, test sample, bead, or yarn made of a material known in the art of bioindicators for carrying test microorganisms during sterilization), or an article containing the aforementioned genetically modified test microorganism disposed thereon or therein (e.g., a strip, test sample, bead, or yarn).

[0114] The method includes positioning a sterilization process bioindicator in a sterilization chamber. The bioindicator has on or contains a plurality of genetically modified test microorganisms, each of which contains a sporulating genetically modified test microorganism. The genetically modified test microorganism is a test microorganism containing a functional fusion gene encoding a non-naturally occurring chimeric protein as described herein. The chimeric protein comprises a first segment and a second segment adjacent to the first segment. The first segment contains at least a portion of a first polypeptide normally present in spores, and the second segment contains a second polypeptide having detectable enzymatic activity, both as described herein.

[0115] When the indicator is positioned in a sterilization chamber, the method includes the step of exposing the biological indicator to a sterilizing agent gas (e.g., vapor, ethylene oxide, hydrogen peroxide vapor, ozone) for a period of time. Exposing the biological indicator to the sterilizing agent gas includes placing a genetically modified test microorganism in contact with the sterilizing agent gas (e.g., vapor contact).

[0116] After exposing the bioindicator to a sterilizing agent gas, the method includes the step of contacting a genetically modified test microorganism with a liquid culture medium and an enzyme substrate capable of reacting with enzyme activity to form a detectable product. According to this disclosure, those skilled in the art will identify a suitable enzyme substrate based on the enzyme activity of a second polypeptide of a chimeric protein. In some embodiments (e.g., in a fully kitted bioindicator), the genetically modified test microorganism, liquid culture medium, and enzyme substrate may co-occur within the housing when the bioindicator is exposed to a sterilizing agent gas. In some embodiments of the method, the enzyme substrate may be disposed in the housing as a dry powder separate from the liquid culture medium (e.g., the liquid culture medium may be disposed in an openable container within the housing). In some embodiments of the method, the enzyme substrate is dissolved and / or suspended in the liquid culture medium co-occupied with the genetically modified test microorganism within the housing.

[0117] In some embodiments, contacting the genetically modified test microorganism with the liquid culture medium includes adding the liquid culture medium (e.g., via a pipette) to a shell or other container in which the genetically modified test microorganism is disposed. In some embodiments, contacting the genetically modified test microorganism with the liquid culture medium includes activating a bioindicator (e.g., by opening a fragile container containing the culture medium in the bioindicator) to induce contact between the culture medium, the enzyme substrate, and the genetically modified test microorganism.

[0118] In some alternative embodiments of this method, the biological indicator may consist only of a substrate (e.g., a plastic film or a glass slide) optionally disposed within a housing (e.g., a tube, cuvette, or microwell). In these embodiments, the biological indicator may need to be placed in a suitable container (e.g., a tube, cuvette, microwell) to which liquid culture medium is added (e.g., via a pipette). Optionally, the enzyme substrate may be added separately to the container, or it may be dissolved and / or suspended in the liquid culture medium added to the container.

[0119] Optionally, after contacting the genetically modified test microorganism with a liquid culture medium and an enzyme substrate capable of reacting with enzyme activity to form a detectable product, the resulting composition comprising the genetically modified test microorganism, the liquid culture medium, and the enzyme substrate may be mixed together (e.g., by manual or mechanical stirring or vortex mixing).

[0120] After contacting the genetically modified test microorganism with a liquid culture medium and an enzyme substrate, the method includes incubating the indicator at a predetermined temperature for a period of time sufficient to detect the presence of a reaction product catalyzed by the enzymatic activity of the chimeric protein. The predetermined temperature can be any incubation temperature suitable for promoting the reaction between the enzyme activity and the enzyme substrate (e.g., a temperature between 25°C and 65°C). The time period can be any suitable incubation period known for detecting enzyme reactions. In some embodiments, the specified time period is less than 8 hours, in some embodiments less than 1 hour, in some embodiments less than 30 minutes, in some embodiments less than 15 minutes, in some embodiments less than 5 minutes, and in some embodiments less than 1 minute. In other embodiments, suitable incubation times for the biological indicator of this disclosure are 10 minutes to 4 hours, or 10 minutes to 1 hour, or 10 minutes to 50 minutes, or 10 minutes to 30 minutes, or 10 minutes to 20 minutes, or 10 minutes to 25 minutes, or 15 minutes to 30 minutes, or 15 minutes to 25 minutes, or 15 minutes to 20 minutes.

[0121] During and / or after the incubation period of the mixture, the products of the enzyme reaction can be detected using procedures known in the art, including visual detection and / or automated detection. Detecting the fluorescent (or colored) products of the enzyme reaction includes, for example, directing electromagnetic radiation (e.g., radiation within the ultraviolet or visible spectrum of electromagnetic energy) into the mixture and detecting the electromagnetic radiation emitted by the fluorescent products in the mixture (e.g., radiation within the ultraviolet or visible spectrum of electromagnetic energy), as described herein. In some embodiments, detecting the electromagnetic radiation emitted by the fluorescent products includes detecting the electromagnetic radiation using an automated detector (e.g., an automated reader as described herein).

[0122] In any embodiment of this method, detecting the fluorescent (or colored) product of the enzyme reaction includes detecting the amount of fluorescence (or color) emitted (or absorbed) by the product of the enzyme reaction. In any embodiment, the amount of fluorescence (or color) detected may be compared to a threshold amount. In any embodiment, a first amount of fluorescence (or color) detected after a first specified time period may be compared to a second amount of fluorescence (or color) detected after a second specified time period. In some embodiments, detecting at least a threshold amount of the enzyme reaction product indicates a lack of efficiency in the sterilization process.

[0123] In any embodiment of the method, positioning the sterilization process bioindicator in the sterilization chamber may include positioning a sterilization process bioindicator comprising a shell in which a genetically modified test microorganism is contained. In these embodiments, contacting the genetically modified test microorganism with a liquid culture medium includes contacting the genetically modified test microorganism with a liquid culture medium inside the shell. In these embodiments, contacting the genetically modified test microorganism with a liquid culture medium may include contacting the genetically modified test microorganism with an aqueous liquid culture medium containing a detection reagent (e.g., a fluorescent or chromogenic enzyme substrate capable of reacting with the enzymatic activity of the chimeric protein).

[0124] In any embodiment of this method, detecting the product includes measuring a parameter associated with the product to obtain a first value. To detect detectable changes caused by a live, genetically modified test microorganism, a bioindicator can be measured immediately after the combination of the liquid culture medium and the genetically modified test microorganism to obtain a first value (e.g., a baseline reading). Subsequently, a second value showing any detectable change deviating from the baseline reading can be detected. The bioindicator can be monitored and measured continuously or intermittently to observe the first and second values. In some embodiments, part or all of the incubation step can be performed before measuring the detectable change.

[0125] In any implementation of this method, the measurement parameters include measuring the emission or absorption of electromagnetic radiation.

[0126] In some embodiments, incubating the bioindicator at a predetermined temperature includes incubating the bioindicator at a temperature between room temperature (e.g., about 23 degrees Celsius) and 70 degrees Celsius. In some embodiments, incubation may be performed at one temperature (e.g., at 37°C, at 50°C-60°C, etc.), while the measurement of the detectable change may be performed at another different temperature (e.g., at room temperature, 25°C, or 37°C). In other embodiments, incubation and the measurement of fluorescence (or color) of the enzyme reaction product are performed at the same temperature.

[0127] The readout time of the bioindicator (i.e., the time used to determine the effectiveness of the sterilization process) may be less than 8 hours in some embodiments, less than 1 hour in some embodiments, less than 30 minutes in some embodiments, less than 15 minutes in some embodiments, less than 5 minutes in some embodiments, and less than 1 minute in some embodiments. In other embodiments, the readout time of the bioindicator of this disclosure is 10 minutes to 1 hour, or 10 minutes to 50 minutes, or 10 minutes to 30 minutes, or 10 minutes to 20 minutes, or 10 minutes to 25 minutes, or 15 minutes to 30 minutes, or 15 minutes to 25 minutes, or 15 minutes to 20 minutes. The detection of fluorescence (or color) above baseline reading indicating the presence of viable spores (i.e., sterilization process failure) may be performed according to any method known in the art, including area under the curve (in a graph of time versus fluorescence (or color) intensity), changes in the slope of the monitoring curve, the use of a fluorescence (or color) threshold, or a combination of two or more of these techniques.

[0128] Reagent test kit

[0129] In another aspect, this disclosure provides a kit for determining the efficacy of a sterilization process. The kit comprises a plurality of genetically modified test microorganisms, each of which comprises a sporulating genetically modified test microorganism; wherein the genetically modified test microorganism comprises a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment; wherein the first segment comprises at least a portion of a first polypeptide normally present in spores; wherein the second segment comprises a second polypeptide having detectable enzyme activity. The kit also comprises an enzyme substrate for detecting enzyme activity. In any embodiment, the kit further comprises instructions for using genetically modified test microorganisms to evaluate the efficacy of a sterilization process.

[0130] In any embodiment, the kit further includes a housing having at least one wall forming an opening to a compartment, as described above. In any embodiment, the housing is sized to accommodate the test microorganism and / or enzyme substrate. In any embodiment, the genetically modified test microorganism and / or enzyme substrate is disposed within the housing.

[0131] In any embodiment of the kit, the genetically modified test microorganism belongs to a genera selected from Bacillus, Geobacillus, Clostridium, and any combination of two or more of the aforementioned genera. In any embodiment, the kit also includes a liquid culture medium suitable for dissolving or suspending the test microorganism and the enzyme substrate, which reacts with the enzymatic activity of the chimeric protein to form a detectable product.

[0132] The invention is illustrated by the following examples. It should be understood that the specific examples, materials, quantities, and processes are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0133] Example

[0134] Example 1. Preparation of a complete set of biological indicators.

[0135] Construction of a shuttle vector containing the gene expressing the fusion protein. *Bacillus stearothermophilus* (ATCC 7953) cells were propagated in tryptone soybean broth (TSB) or tryptone soybean agar (TSA) at 60°C. *Escherichia coli* (DH5a) was propagated in Luria broth (LB) at 37°C. The plasmid pJet1.2 was an *E. coli* / Bacillus shuttle vector obtained from Addgene.org (Watertown, MA). The plasmid pJet1.2 contains genetic markers for ampicillin selection in *E. coli* transformed with pJet1.2 and for chloramphenicol resistance in *Bacillus* species transformed with pJet1.2. See also Figure 1 This figure shows the annotated gene map of pJet1.2.

[0136] The fusion gene construct shown in SEQ ID NO.1 was synthesized by Genescript (Piscataway, NJ). The construct includes the small acid-soluble protein (SASP) gene sspL from *Bacillus thermophilus*. It also includes the promoter region of the sspL gene from the *Bacillus thermophilus* genome. The construct was formed by in-frame fusion of a portion of the N-terminus of the sspL gene with an α-glucosidase gene from *Bacillus thermophilus*. A linker peptide coding region is positioned between the sspL coding portion and the α-glucosidase coding portion of the fusion gene construct. PstI and BbsI restriction enzyme sites were added to the N-terminus and C-terminus of the construct, respectively, and used to insert the fusion gene into plasmid pJet1.2.

[0137] Following the restriction enzyme supplier's protocol, the fusion gene construct and pJet1.2 plasmid were digested with PstI and BbsI. The digested molecules were then ligated together, resulting in the insertion of the fusion gene construct into the PstI and BbsI sites in pJet1.2. The resulting plasmid was then transformed into *E. coli* DH5α, replicated, and the DNA was purified using a plasmid micropreparation kit. The purified plasmid DNA was sequenced to ensure it contained the fusion insert. The plasmid was then first transformed into *Bacillus subtilis*, then purified again from *Bacillus subtilis* and transformed into *Bacillus stearothermophilus*. Transformants (genetically modified test microbes) were selected on TSA agar containing chloramphenicol. Chloramphenicol-resistant colonies were then picked to obtain genetically modified *Bacillus stearothermophilus* test microbes.

[0138] Construction of the bioindicator. Genetically modified test microbial spores of *Bacillus stearothermophilus* were generated using chloramphenicol added to agar spore-forming medium. Spores were harvested from agar plates and purified by washing several times with sterile deionized H₂O. The spores were then coated onto a polypropylene membrane. The coated carrier was dried, and the dye was cut into individual spore carriers (approximately 2 mm × 4 mm).

[0139] 3M TM ATTEST TM The ultra-fast readout bioindicator (part number 1492V) is from 3M Corporation (St. Paul, MN). Articles of manufacture similar to those bioindicators are described in U.S. Patent Application Publication No. 2014 / 0349335, the entire contents of which are incorporated herein by reference. The cap of the 1492V bioindicator is removed, and the contents (glass ampoule, insert, and spore reservoir) are removed and reassembled in the same manner, except that the spore reservoir of the commercially available bioindicator is replaced with a spore carrier coated with spores of the aforementioned genetically modified test microorganism.

[0140] Example 2. Detection of chimeric protein enzyme activity in a modular bioindicator exposed to steam sterilizing agents. sex .

[0141] In an AMSCO Model 3013 automated sterilizer (Steris Corporation, Mentor, OH), ten assembled, fully-equipped bioindicators of Example 1 were exposed to a pre-vacuum steam sterilization cycle at 132.2°C. The length of exposure to the 132.2°C steam was selected such that most spores in each bioindicator were killed, but at least one spore was expected to survive in approximately 10% to approximately 90% of the bioindicators (i.e., a process referred to in the art as a “partial cycle”). The bioindicators containing genetically modified *Bacillus stearothermophilus* spores were monitored using an automated bioindicator fluorescence reader from 3M (St. Paul, MN). Fluorescent detection of α-glucosidase activity was observed in the bioindicators, with at least one spore surviving upon exposure to the steam sterilizing agent.

[0142] Example 3. Preparation of a complete set of biological indicators .

[0143] *Bacillus stearothermophilus* (ATCC 7953) was propagated and grown at 60°C in tryptone soybean broth (TSB) or on tryptone soybean agar (TSA). *Escherichia coli* (DH5α) was propagated in Luria broth (LB) at 37°C. pSTE12 is an *E. coli* / Bacillus shuttle vector containing genetic markers for ampicillin selection in *E. coli* and tetracycline resistance in *Bacillus stearothermophilus*. pSTE12 includes ampicillin resistance (Amp... r ) and tetracycline resistance (Tc r Genes, origin of replication (ori), and β-galactosidase genes, including multiple cloning sites. See also Figure 2 Comments on pSTE12.

[0144] The fusion construct shown in SEQ ID NO.2 was synthesized by GenScript. This construct includes the small acid-soluble protein (SASP) gene sspL from *Bacillus thermophilus*. It also includes the promoter region of the sspL gene from the *Bacillus thermophilus* genome. The construct was formed by in-frame fusion of a portion of the N-terminus of the sspL gene with an α-glucosidase gene from *Bacillus thermophilus*. KpnI and EcoRI restriction enzyme sites were added to the N-terminus and C-terminus of the construct, respectively, for insertion into the multiple cloning site of pSTE12.

[0145] The pyrF gene from a Bacillus species encodes an orotic nucleoside 5'-phosphate decarboxylase orthoside from the eukaryotic ura3 gene and is essential for pyrimidine biosynthesis and the metabolism of 5-fluoroorthoic acid (5-FOA) into toxic metabolites. Disruption of pyrF in *Bacillus thermophilus* leads to uracil auxotrophy and resistance to 5-FOA. An integrative plasmid was designed for single-exchange homologous recombination, containing a region homologous to pyrF, including a ~400 bp upstream sequence. The pyrF gene was synthesized by GenScript and includes flanking HindIII and PstI restriction sites for insertion into the multiple cloning site of pSTE12.

[0146] A plasmid construct containing the fusion gene of SEQ ID NO.2 and the pyrF gene from *Bacillus thermophilus* was introduced into *Bacillus thermophilus* via protoplast transformation or binding transfer. Transformants were selected for resistance to 5-FOA in the presence of uracil, and then cultured on agar plates with or without tetracycline.

[0147] Tetracycline-resistant colonies were then selected to produce *Bacillus stearothermophilus* spores in or on a spore-forming medium containing tetracycline. The spores were then purified by washing several times with dH₂O. The spores were then coated onto a polypropylene membrane. The coated carrier was dried, and the dye was cut into individual carriers (approximately 2 mm × 4 mm). The carriers were then assembled into a complete kit bioindicator as described in Example 1.

[0148] Example 4. Detection of chimeric protein enzyme activity in a modular bioindicator exposed to steam sterilizing agents. sex .

[0149] Then, in an AMSCO Model 3013 automated sterilizer, the assembled BI, including genetically engineered spores with fusion constructs, was exposed to a pre-vacuum steam sterilization cycle at 132.2°C. When compared with the same bioindicator construct containing native *Bacillus stearothermophilus* spores, at any given exposure time, the bioindicator with genetically engineered spores resulted in faster fluorescence detection of 4-methylumbelliferone-α-D-glucoside (4MUG) relative to pH color change growth response.

[0150] While the numerical ranges and parameters shown within the broad scope of this invention are approximate, the values ​​shown in specific embodiments are reported as accurately as possible. Nevertheless, standard deviations arising during the respective testing processes inevitably result in all values ​​inherently including a certain range.

[0151] All headings are for the reader's convenience and should not be used to limit the meaning of the text that follows the heading, unless otherwise specified.

[0152] All patents, patent applications, publications, and the full disclosure of provisions of nucleic acid and protein databases cited herein are incorporated herein by reference as if each were individually incorporated. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention, and it should be understood that the invention is not unduly limited to the exemplary embodiments presented herein.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] sequence list <110> 3M Innovative Properties Company <120> Bioindicators with modified test microorganisms <130> 82825US002 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 2078 <212> DNA <213> Thermophilic Bacillus stearothermophilus <220> <221> misc_structure <222> (1)..(6) <223> PstI restriction sites <220> <221> misc_signal <222> (157) (159) <223> start codon <220> <221> misc_structure <222> (157) (489) <223> The coding sequence of the truncated sspL gene from Bacillus stearothermophilus <220> <221> misc_structure <222> (490)..(2072) <223> Coding sequence of α-glucosidase. <220> <221> misc_signal <222> (2070)..(2072) <223> Stop codon <220> <221> misc_structure <222> (2073)..(2078) <223> BbsI restriction site <400> 1 ctgcagaatg gctgcatcaa acttcgcaaa tttaacataa tacatagtac agtcagcagc 60 cattgggcac agccgagaag gcagtgactc cccggattca cggctggtgc aactccagca 120 agcccaacca tactatatat aaaaggagat ataacaatgg ctgcatcaaa cttcgcaaat 180 ttaacataat acatagtaca gtcagcagcc attgggcaca gccgagaagg cagtgactcc 240 ccggattcac ggctggtgca actccagcaa gcccaaccat actatatata aaaggagata 300 taacaatgcc aaaccaatct ggaagtaact cttcaaacca actacttgta cctggcgcag 360 ctcaggtaat cgatcaaatg aaattcgaag gcggcggcgg cagcttgaaa aaaacatggt 420 ggaaagaggg cgttgcgtat caaatttatc ctcgcagctt tatggatgcc aacggcgacg 480 gcatcggtga tcttcgcggc atcatcgaaa agctggatta tttggtggag cttggagtcg 540 acatcgtttg gatttgtccg atttaccggt cgccgaatgc cgataacgga tatgacatca 600 gcgattatta tgccattatg gatgagtttg gaacgatgga tgacttcgat gaattgcttg 660 cccaagccca tcggcgcggg ttgaaaatca ttttggattt ggtcatcaat catacgagtg 720 atgagcatcc gtggtttatc gaatcgcggt catcgcgaga caatccgaag cgcgactggt 780 acatttggcg cgacggcaaa gacgggcgcg aaccgaacaa ctgggaaagc attttcggcg 840 gctcggcatg gcagtatgac gagcggacgg gtcagtatta cttgcatatt tttgacgtca 900 aacagcccga cttgaattgg gaaaacgacg aagtgcggcg ggcgctttat gcgatgatca 960 actggtggct ggataaaggc atcgacggct ttcgcatcga cgcgatttcc cacattaaga 1020 aaaagccggg tcttcccgat ttgccaaatc cgaaggggct gaagtacgtg ccgtcatttg 1080 ctgcgcacat gaaccagcca gggattatgg agtatttgcg agagttgaaa gagcaaacgt 1140 ttgcacgata tgacattatg acggtcggcg aggcgaacgg agtaacggtt gatgaggccg 1200 aaaatgggt cgggggaaa aacggcgtgt ttcatatgat ttttcagttt gagcatttag 1260 ggctttggaa aaggaaagcc gatggttcga tcgatgtccg ccggctgaag cggacgttga 1320 1380 acttgcctcg atcagtgtcg acatgggggaa atgacggcga gtattgggcg gagagcgcga 1440 aggcgcttgg cgcgctctac tttttcatgc aagggacgcc gttcatttac caagggcaag 1500 agatcgggat gacgaacgtg caattctccg acattcgcga ctaccgcgat gtcgctgcct 1560 tgcgtctgta tgaactcgaa cgggcgaacg gccggacaca tgaggaagtg atgaagatca 1620 tttggcaaac cgggcgcgac aactcgcgca ccccgatgca atggtctggt gccccaaacg 1680 ctggattcac gacaggtacg ccatggatca aggtgaacga aaactatcgt acgatcaatg 1740 tcgaggccga gcggcgcgac ccgaactcgg tatggtcgtt ttatcgacaa atgattcagc 1800 ttcggaaagc gaacgagctg tttgtttatg gaacgtacga tctgcttttg gaaaaccacc 1860 catccattta cgcgtacaca agaacgcttg gccgcgatcg ggcgcttatc attgtcaacg 1920 tatccgatcg tccttcactt taccgctatg acggcttccg ccttcagtca agcgatttgg 1980 cgctctcgaa ctacccggtc cgtccgcata aaaatgcgac gcgttttaag ctgaagccgt 2040 acgaggcgcg tgtatacatc tggaaagaat aagaagac 2078 <210> 2 <211> 2063 <212> DNA <213> Thermophilic Bacillus stearothermophilus <220> <221> misc_structure <222> (1)..(6) <223> KpnI restriction sites <220> <221> misc_structure <222> (157) (159) <223> start codon <220> <221> misc_structure <222> (157) (391) <223> The truncated coding sequence of the sspL gene <220> <221> misc_structure <222> (392)..(2057) <223> The coding sequence of the α-glucosidase gene <220> <221> misc_signal <222> (2055)...(2057) <223> stop codon <220> <221> misc_structure <222> (2058)...(2063) <223> EcoRI restricted sites <400> 2 ggtaccaatg gctgcatcaa acttcgcaaa tttaacataa tacatagtac agtcagcagc 60 cattgggcac agccgagaag gcagtgactc cccggattca cggctggtgc aactccagca 120 agcccaacca tactatatat aaaaggagat ataacaatgg ctgcatcaaa cttcgcaaat 180 ttaacataat acatagtaca gtcagcagcc attgggcaca gccgagaagg cagtgactcc 240 ccggattcac ggctggtgca actccagcaa gcccaaccat actatatata aaaggagata 300 taacaatgcc aaaccaatct ggaagtaact cttcaaacca actacttgta cctggcgcag 360 ctcaggtaat cgatcaaatg aaattcgaaa tgaaaaaaac atggtggaaa gagggcgttg 420 cgtatcaaat ttatcctcgc agctttatgg atgccaacgg cgacggcatc ggtgatcttc 480 gcggcatcat cgaaaagctg gattatttgg tggagcttgg agtcgacatc gtttggatttt 540 gtccgattta ccggtcgccg aatgccgata acggatatga catcagcgat tattatgcca 600 tttggatga gtttggaacg atggatgact tcgatgaatt gcttgcccaa gcccatcggc 660 gcgggttgaa aatcatttg gatttggtca tcaatcatac gagtgatgag catccgtggt 720 ttatcgaatc gcggtcatcg cgagacaatc cgaagcgcga ctggtacatt tggcgcgacg 780 gcaaagacgg gcgcgaaccg aacaactggg aaagcatttt cggcggctcg gcatggcagt 840 atgacgagcg gacgggtcag tattacttgc atatttttga cgtcaaaac cccgacttga 900 attgggaaaa cgacgaagtg cggcgggcgc tttatgcgat gatcaactgg tggctggata 960 aaggcatcga cggcttcgc atcgacgcga tttcccacat taagaaaaag ccgggtcttc 1020 ccgatttgcc aaatccgaag gggctgaagt acgtgccgtc atttgctcg cacatgaacc 1080 agccagggat tatggagtat ttgcgagagt tgaaagagca aacgtttgca cgatatgaca 1140 ttatgacggt cggcgaggcg aacggagtaa cggttgatga ggccgaacaa tgggtcgggg 1200 aagaaaacgg cgtgtttcat atgatttttc agtttgagca tttagggctt tggaaagga 1260 aagccgatgg ttcgatcgat gtccgccggc tgaagcggac gttgacgaaa tggcaaaaag 1320 gattggaaaa ccgtgggtgg aacgcgctct ttttggaaaa ccacgacttg cctcgatcag 1380 tgtcgacatg gggaaatgac ggcgagtatt gggcggagag cgcgaaggcg cttggcgcgc 1440 tctacttttt catgcaaggg acgccgttca tttaccaagg gcaagagatc gggatgacga 1500 acgtgcaatt ctccgacatt cgcgactacc gcgatgtcgc tgccttgcgt ctgtatgaac 1560 tcgaacgggc gaacggccgg acacatgagg aagtgatgaa gatcatttgg caaaccgggc 1620 gcgacaactc gcgcaccccg atgcaatggt ctggtgcccc aaacgctgga ttcacgacag 1680 gtacgccatg gatcaaggtg aacgaaaact atcgtacgat caatgtcgag gccgagcggc 1740 gcgacccgaa ctcggtatgg tcgttttatc gacaaatgat tcagcttcgg aaagcgaacg 1800 agctgtttgt tttggaacg tacgatctgc ttttggaaaa ccacccatcc atttacgcgt 1860 acacaagaac gcttggccgc gatcgggcgc ttatcattgt caacgtatcc gatcgtcctt 1920 cacttaccg ctatgacggc ttccgccttc agtcaagcga tttggcgctc tcgaactacc 1980 cggtccgtcc gcataaaaat gcgacgcgtt ttaagctgaa gccgtacgag gcgcgtgtat 2040 acatctggaa agaataagaa ttc 2063 <210> 3 <211> 1137 <212> DNA <213> Read more <220> <221> misc_structure <222> (1)..(6) <223> HindIII restriction site <220> <221> misc_structure <222> (488)..(490) <223> start codon of pyrF gene <220> <221> misc_structure <222> (1131)..(1137) <223> PstI restriction site <400> 3 aagcttacgt ggctaatatc acagaaattg cattagcgat cgaggaagcg ggagcggacg 60 gtcttacgat gatcaacaca ctaatcggca tgagactcga tttaaaaacc ggcaaaccga 120 tattagcgaa taaaacaggg ggactttcgg gccctgctgt gaagccggtt gccattcgca 180 tggtgtatga agtcagccag atggtcaaca tcccgattat cggaatggga ggcgtgcaaa 240 cggctgaaga tgccctggaa tttcttctcg cgggagcaag cgcagtcgct gtcggaacag 300 caaactttgt gaatcctttt gcatgtccag agattattga acagctccca tctgttttgc 360 tccaatacgg ctatcaatca attgaagaat gcatcggaag gagctggaat catgaaaaac 420 aacctgccca tcatcgcgct tgattttgcg tcagctgaag aaacacttgc gttcttagcg 480 ccttttcagc agaaccgtt atttgtaag gttgggatgg agcttttta tcaagaaggg 540 ccatctatcg tgaacaact aaagaaaga aactgcgagc tattttaga tctaaagctt 600 catgacatcc cgactactgt aaaaaagcg atgaagcgcc ttgccagtct tggagtagac 660 ctcgtcaatg ttcatgctgc cgggggcaaa aaatgatgc aggcagctct cgaggctta 720 gagaaggta cgccggctgg aaaaaaacgt ccgtcactta tcgcggtaac ccagctgaca 780 agcacatctg aaaatcat gaagatgaa ctgctgatcg aaagtctct gattgatcg 840 gttgtgcact acagcaaaca ggcggaagaa agcggactgg atggagtgt ctgctctgtt 900 catgaagcaa aagccatta ccaagcggtg tcgccttcat ttctgactgt cactccgggg 960 atcagaatgt cagaggacgc tgcgaatgac caagttcgcg tagcgacgcc tgccattgca 1020 aggagaaag gttcatcagc gattgtagta ggacgctcga ttacaaagc ggaagacccg 1080 gtaaaagcct aaggctgt cagacttgaa tgggagga tcaatcttg aggtacc 1137

Claims

1. An article for evaluating the effectiveness of a sterilization process, said article comprising: A housing having at least one wall forming an opening to a compartment; A plurality of genetically modified test microorganisms are disposed in the shell, each of the plurality of genetically modified test microorganisms comprising a sporulating genetically modified test microorganism; The genetically modified test microorganism contains a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment; The first segment contains at least a portion of a first polypeptide that is normally present in spores; The second segment contains a second polypeptide with detectable enzyme activity; A liquid culture medium disposed in an openable container, the openable container being disposed within or attached to the housing; as well as An enzyme substrate, which is capable of reacting with the detectable enzyme activity to form a detectable product, wherein the enzyme substrate is disposed in the housing or the openable container; The first polypeptide normally present in the spore contains at least a portion of a first polypeptide segment comprising not less than 1% of the amino acid residues constituting the first polypeptide.

2. The article of claim 1, wherein the first polypeptide is selected from small acid-soluble spore protein, spore coat protein and spore formation-associated GTP-binding protein.

3. The article of claim 2, wherein the first polypeptide is selected from... ysxE , yutH , cotE , cotF , cotH , cotS , cotF , gerE , sspA , sspB , sspD and sspE The gene encoding.

4. The article according to claim 1, wherein the second polypeptide has enzymatic activity selected from esterase, lipase, glycosidase, aminopeptidase, phosphatase, and luciferase.

5. The article of claim 4, wherein the second polypeptide has an enzymatic activity selected from the group consisting of: β-D-galactosidase; β-D-glucosidase; α-D-glucosidase; alkaline phosphatase; acid phosphatase; butyrate esterase; caprylate esterase; chloramphenicol acetyltransferase; catechol-2,3-dioxygenase; myristate lipase; leucine aminopeptidase; valine aminopeptidase; chymotrypsin; phosphorylhydrolase; α-D-galactosidase; α-L-arabinofuranase; N-acetyl-β-aminoglucosidase; β-D-cellobiosidase; alanine aminopeptidase; proline aminopeptidase; tyrosine aminopeptidase; phenylalanine aminopeptidase; β-D-glucuronidase; and fatty acid esterase.

6. The article of manufacture according to any one of claims 1 to 5, wherein the first segment comprises the N-terminal region of the first polypeptide.

7. The article of manufacture according to any one of claims 1 to 5, wherein the functional fusion gene comprises at least one regulatory sequence controlling the expression of the first polypeptide.

8. The article of manufacture according to any one of claims 1 to 5, wherein the genetically modified test microorganism belongs to the genus Bacillus (Bacillus). Bacillus ), Bacillus spp. Geobacillus Clostridium ( Clostridium ) and any combination of two or more of the aforementioned genera.

9. The article of claim 1 to 5, wherein the liquid culture medium comprises an enzyme substrate dissolved and / or suspended therein.

10. The article of manufacture according to any one of claims 1 to 5, wherein the fusion gene is located in an extrachromosomal replicon in the genetically modified test microorganism.

11. The article of manufacture according to any one of claims 1 to 5, wherein the fusion gene is located in a chromosome replicon in the genetically modified test microorganism.

12. A method for determining the effectiveness of a sterilization process, the method comprising: The biological indicator for the sterilization process is positioned in the sterilization chamber; The bioindicator has or contains a plurality of genetically modified test microorganisms, each of the plurality of genetically modified test microorganisms comprising a sporulating genetically modified test microorganism; The genetically modified test microorganism contains a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment; The first segment contains at least a portion of a first polypeptide that is normally present in spores; The second segment contains a second polypeptide with detectable enzyme activity; When the indicator is positioned in the sterilization chamber, the biological indicator is exposed to sterilizing agent gas; After exposing the bioindicator to the sterilizing agent gas, the test microorganism is brought into contact with a liquid culture medium and an enzyme substrate capable of reacting with the enzyme activity to form a detectable product; After contacting the genetically modified test microorganism with the liquid culture medium and enzyme substrate, the biological indicator is incubated at a predetermined temperature for a period of time. as well as The product in the liquid culture medium was detected; Detecting the product includes measuring a parameter associated with the product to obtain a first value.

13. The method of claim 12, wherein positioning the sterilization process bioindicator in the sterilization chamber comprises positioning a sterilization process bioindicator comprising a housing having the test microorganism therein, wherein contacting the test microorganism with the liquid culture medium comprises contacting the test microorganism with the liquid culture medium inside the housing.

14. The method of claim 13, wherein contacting the test microorganism with the liquid culture medium comprises contacting the test microorganism with an aqueous culture medium containing the enzyme substrate.

15. The method according to any one of claims 12 to 14, wherein incubating the bioindicator at a predetermined temperature comprises incubating the bioindicator at a temperature between 23 degrees Celsius and 70 degrees Celsius.

16. The method according to any one of claims 12 to 14, wherein the measurement parameters include measuring the emission or absorption of electromagnetic radiation.

17. The method of claim 16, wherein the method further comprises comparing the first value with a second value.

18. A reagent kit comprising: A plurality of genetically modified test microorganisms, each of the plurality of genetically modified test microorganisms comprising a sporulating genetically modified test microorganism; The genetically modified test microorganism contains a functional fusion gene encoding a non-naturally occurring chimeric protein, the chimeric protein comprising a first segment and a second segment adjacent to the first segment; The first segment contains at least a portion of a first polypeptide normally present in spores, the portion containing a first polypeptide segment comprising not less than 1% of the amino acid residues constituting the first polypeptide; The second segment contains a second polypeptide with detectable enzyme activity; Enzyme substrates used for the detectable enzyme activity; as well as A liquid culture medium suitable for dissolving or suspending the test microorganism and the enzyme substrate, wherein the enzyme substrate is capable of reacting with the enzyme activity of the chimeric protein to form a detectable product.

19. The kit of claim 18, further comprising a housing having at least one wall forming an opening to a compartment.

20. The kit of claim 19, wherein the size of the housing is designed to accommodate the test microorganism and / or the enzyme substrate.

21. The kit of claim 20, wherein the genetically modified test microorganism and / or the enzyme substrate are disposed in the housing.

22. The kit according to any one of claims 18 to 21, wherein the genetically modified test microorganism belongs to the genera selected from: Bacillus, Geobacillus, Clostridium, and any combination of two or more of the foregoing genera.

23. The kit according to any one of claims 18 to 21, wherein the genetically modified test microorganism is mounted on a vector.

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