WO2018071732A1

WO2018071732A1 - Systems and methods for sterility assurance

Info

Publication number: WO2018071732A1
Application number: PCT/US2017/056424
Authority: WO
Inventors: Brigitte ANGENIUEX; Joshua Palmer; Edward Perkins
Original assignee: Verrix, Llc
Priority date: 2016-10-12
Filing date: 2017-10-12
Publication date: 2018-04-19

Abstract

A system for testing the efficacy of a sterilization process includes a bioindicator housing a genetically modified test microorganism modified with a heterologous reporter gene, and a detection apparatus for detecting the presence or absence of an optically detectable signal from the heterologous reporter gene. A method of testing the efficacy of a sterilization process includes subjecting the bioindicator to the sterilization process, introducing an expression composition to the genetically modified test microorganism after completion of the sterilization process, and detecting the presence or absence of the optically detectable signal. The presence of the optically detectable signal indicates the failure of the sterilization process, while the absence of the optically detectable signal indicates a successful sterilization process.

Description

SYSTEMS AND METHODS FOR STERILITY ASSURANCE CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The present application claims priority to and the benefit of U.S. Provisional Application Serial No. 62/407,434 filed on October 12, 2016, entitled "Bioindicator Organism and Assay," the entire content of which is incorporated herein by reference. BACKGROUND

[0002] Biological indicators are packages or containers used to test the efficacy of sterilization processes. Biological indicators typically include a microorganism that is resistant to the sterilization process being tested and a culture medium housed within a package or container. Current sterility assurance technologies utilize assays that require at least one day for direct (and at least 20 minutes for indirect) measurements of microorganism survival within a biological indicator (or "bioindicator") that has been subjected to the sterilization process being tested. Most of the assays currently used to determine microorganism survival within the bioindicator rely on indirect

measurement of microorganism survival and do not quantify the microorganism survival. Additionally, current sterility assurance technologies often rely on these non- quantitative measurements of microorganism survival, and simply return a positive result (indicating microorganism survival and therefore sterilization failure) or a negative result (indicating no detected microorganism survival and therefore

sterilization success).

SUMMARY

[0003] Embodiments of the present invention are directed to sterility assurance and bioburden monitoring. Embodiments of the present invention include bioindicators, and bioindicator detection systems and methods that utilize a genetically modified bioindicator test organism incorporated into the bioindicator, e.g., a self-contained bioindicator package or container. The bioindicator test microorganism in the bioindicator is an organism that has a known resistance to sterilization, and in some embodiments, is an organism that has a known resistance to the particular sterilization process that is being tested. According to embodiments of the present invention, the test microorganism in the bioindicator device is genetically modified to qualitatively or quantitatively enhance or change detection. For example, in some embodiments, the test microorganism may be genetically modified to enable more rapid and direct detection, or to enable quantitative measurement of the viability of the organism. In some embodiments, the detection is by means of a signal that is detectable as a change in the luminescence, fluorescence, phosphorescence, absorbance, refractivity, or optical activity of the test microorganism in the bioindicator.

[0004] In some embodiments of the present invention, a method for testing the efficacy of a sterilization process includes identifying the viability of a genetically modified test microorganism (i.e., an organism known to have some level of resistance to sterilization, or to the particular sterilization process) after exposure to the

sterilization process. In some embodiments, identifying the viability of the test microorganism comprises exposing a bioindicator (e.g., a package or a container) that contains the genetically modified test microorganism and a suitable culture medium to the sterilization process, and detecting viability of the test microorganism after the sterilization process has completed. In some embodiments, the method includes positing a genetically modified test microorganism in the bioindicator. The genetically modified test microorganism comprises a test microorganism that has been modified with a heterologous reporter gene capable of producing an optically detectable signal. The method further includes subjecting the bioindicator to the sterilization process. After the sterilization process is complete, the method further includes introducing to the test microorganism in the bioindicator an expression composition capable of inducing expression of the heterologous reporter gene. The genetically modified test microorganism and the expression composition may be allowed to incubate for a suitable period of time, after which the method further comprises identifying the presence or absence of the optically detectable signal. Alternatively, in some embodiments, the incubation period may be omitted, and identification of the presence or absence of the optically detectable signal may be performed immediately or shortly after introduction of the expression medium to the genetically modified test

microorganism. When the optically detectable signal is detected, the method further comprises identifying the test microorganism as viable, and therefore that the sterilization process was unsuccessful. When the optically detectable signal is not detected, the method further comprises identifying the test microorganism as not viable, and therefore that the sterilization process was successful.

[0005] In some embodiments of the present invention, a system for identifying efficacy of a sterilization process includes determining the viability of a genetically modified test microorganism after exposure to the sterilization process. In some embodiments, the system includes a bioindicator including the genetically modified test microorganism housed or contained within a suitable package or container. According to some embodiments, the genetically modified test microorganism includes a test microorganism that is known to have some level of resistance to sterilization (or in some embodiments is resistant or has some level of resistance to the specific sterilization process being tested), and that has a plasmid encoded with a heterologous reporter gene that is capable of producing an optically detectable signal. In some embodiments, the bioindicator further includes an expression composition capable of inducing expression of the heterologous reporter gene. In embodiments in which the expression composition is included within the bioindicator such that the expression composition would be present in the device prior to and during the sterilization process, the bioindicator device is configured to retain the expression composition separate from the genetically modified test organism until after the sterilization process is complete (such as, for example, in a breakable pouch or capsule housed within the container or package of the bioindicator).

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] These and other features and advantages of embodiments of the present disclosure will be better understood and appreciated when considered in conjunction with the accompanying drawings.

[0007] Fig. 1 is a schematic cross-sectional view of a bioindicator device according to embodiments of the disclosure.

[0008] Fig. 2 is schematic cross-sectional view of a bioindicator device with a self- contained germinant or growth media reservoir according to embodiments of the present disclosure.

[0009] Fig. 3 is a schematic of a plasmid construct (used to genetically modify the test microorganism) encoding a reporter gene 8, regulatory elements 6,9, and a selection gene 7, according to embodiments of the present disclosure.

[0010] Fig. 4 is a schematic of a plasmid construct (used to genetically modify the test microorganism) encoding a reporter gene (super folded green fluorescent protein (sfGFP)), regulatory elements (an origin of replication (ORI) and the atpA F0F1 ATP synthase subunit alpha promoter (PatpA)), and an antibiotic resistance (abR) gene, according to embodiments of the present disclosure. [0011] Fig. 5 is a schematic of a plasmid construct (used to genetically modify the test microorganism) encoding a reporter gene (sfGFP), regulatory elements (and ORI and the chemical-inducible promoter (Pspac)), and an antibiotic resistance (abR) gene, according to embodiments of the present disclosure.

[0012] Fig. 6 is a schematic of a plasmid genetic construct (used the genetically modify the test microorganism) encoding a reporter gene (sfGFP), regulatory elements (an ORI and the lactate dehydrogenase gene (Pldh)), and an antibiotic resistance (abR) gene, according to embodiments of the present disclosure.

[0013] Fig. 7 is a schematic plasmid map of plasmid pNW33N as described in the present disclosure.

[0014] Fig. 8 is a schematic plasmid map of plasmid pUC02-AMP-RP1 s1 promoter and fast folder thermal stable yellow fluorescent protein (fftsYFP), including ampR (ampicillin resistance gene) and Notl cloning sites as indicated and described in the present disclosure.

[0015] Fig. 9 is a schematic plasmid map of plasmid pNW33N with RPIsl promoter and fftsYFP gene as described in the present disclosure.

[0016] Figs. 10A and 10B show fluorescent microscopy images of Geobacillus stearothermophilus transfected with pNW33N-fftsYFP plasmid using conjugative plasmid transfer in which Fig. 10A shows fluorescent microscopy using a Semrock brightline LED-YFP-A-NTE filter cube; and Fig. 10B shows phase imaging using a 40x phase objective as described in the present disclosure.

[0017] Figs. 1 1 A and 1 1 B show fluorescent microscopy images of Geobacillus stearothermophilus transfected with pNW33N-fftsYFP plasmid using electroporation in which Fig. 1 1 A shows fluorescent microscopy using a DAPI filter cube and Fig. 1 1 B shows fluorescent microscopy using Semrock Brightline LED-YFP-A-NTE filter cube as described in the present disclosure. [0018] FIG. 12 is a schematic view of an example system for sterility assurance testing partially cut away to show relevant portions of the interior of the depicted detection apparatus, according to embodiments of the present disclosure.

[0019] FIG. 13 is a schematic view of an example system for sterility assurance testing, according to embodiments of the present disclosure.

DETAILED DESCRIPTION

[0020] The present disclosure relates to biological indicators (e.g., containers or packages) and their inclusion in verification systems and methods for verifying the efficacy of a sterilization process. As used herein, the term "biological indicator" may also be referred to interchangeably as "bioindicator" or "Bl." In some embodiments of the present disclosure, a bioindicator is a container or package that houses a

genetically modified test microorganism that has been genetically modified with a heterologous reporter gene. As used herein, the term "genetically modified test microorganism" refers to the genetic modification of the test microorganism with the referenced heterologous reporter gene. Genetic modification of the test microorganism may be carried out by any suitable method known in the art. For example, a plasmid encoding the heterologous reporter gene may be introduced into the microorganism by bacterial transformation, transfection, electroporation, and/or any other means including transduction, nonlimiting examples of which are described in this disclosure. According to embodiments of the present invention, the bioindicator is placed in the sterilization chamber or otherwise exposed to the sterilization process to be tested. After the sterilization process is completed, the bioindicator is removed from the sterilization chamber (or otherwise removed from exposure to the sterilization process), and an expression composition is introduced to the genetically modified test

microorganism. In some embodiments, the expression composition and genetically modified test microorganism are allowed to incubate for a suitable amount of time. In some embodiments, however, the incubation period may be omitted (and, e.g., the bioindicator may be immediately placed in or on a detection apparatus for detection as discussed further below). The expression composition includes a germinant and/or culture medium containing media suitable for growth of the genetically modified test microorganism and expression reagents capable of inducing expression of the heterologous reporter gene. As understood by those skilled in the art, inducing expression of a heterologous reporter gene with regulatory reagents occurs in about 5 minutes or as little as 5 minutes. Accordingly, if a genetically modified test organism survived the sterilization process, upon introduction of the expression composition, the heterologous reporter gene is expressed in approximately 5 minutes. In comparison, some conventional methods of determining microorganism survival or viability require the accumulation of a substrate from an enzymatic reaction in order to detect survival or viability, which process takes at least 20 minutes, and sometimes longer.

Accordingly, the methods and systems according to embodiments of the present invention enable significantly shorter wait times before the success or failure of a sterilization process is determined. Indeed, according to embodiments of the present disclosure, survival or viability of the genetically modified test microorganism is detected upon activation of the cellular machinery resulting in expression of the heterologous reporter gene, which is a significantly shorter time period than that required to accumulate enough substrate (from an enzymatic reaction) for detection.

[0021] After completion of the sterilization process (when no incubation is performed) or after suitable incubation (if performed), the bioindicator is subjected to detection by a detection apparatus (e.g., the bioindicator may be placed in or on the detection apparatus, or is otherwise connected to or capable of interaction with the detection apparatus), which detects (e.g., measures or reads) the viability of the genetically modified microorganism. This detection may be accomplished by detecting, measuring or reading an optically detectable signal emitted by any viable genetically modified test microorganisms remaining in the bioindicator after exposure to the sterilization process. This optically detectable signal is made possible by the introduction (e.g., by transformation) of the genetically modified test microorganism with the heterologous reporter gene, and will only be detectable after the sterilization process has completed if viable genetically modified test microorganisms remain in the bioindicator. Accordingly, if the detection apparatus detects the optically detectable signal (or the optically detectable signal is otherwise detected, for example by observation in the bioindicator by the naked eye), this indicates the presence of viable genetically modified test microorganisms, which in turn, indicates that the sterilization process was unsuccessful. Conversely, if the optically detectable signal is not detected (e.g., either by the detection apparatus or by naked-eye observation), this indicates either that no viable genetically modified test microorganisms remain in the bioindicator device or that any remaining viable genetically modified test

microorganisms are undetectable. In either case, the failure to detect the optically detectable signal indicates that the sterilization process was successful.

[0022] The verification systems and methods disclosed herein may be used to verify any suitable sterilization process. Some known sterilization processes include those using pressurized steam, vaporized hydrogen peroxide or ethylene oxide, and any other common sterilants that are used to sterilize tools, equipment and supplies, for example those used in hospitals and other healthcare facilities. The goal of any sterilization process is to eliminate biological activity from the products undergoing sterilization to prepare them for use in the next procedure (e.g., surgery or other medical procedure), but as the tools, equipment or supplies must remain sterile for use in the next procedure, the tools, equipment and supplies themselves cannot be tested for sterility assurance. Accordingly, a common practice to assure the sterility of these items is to include a bioindicator in each sterilization cycle (or "batch"), and to then test the bioindicator for sterility. Bioindicators typically include a "test" microorganism that is known to have some level of resistance to sterilization or to the specific sterilization process being tested, and sterility assurance testing typically involves detecting whether any viable microorganisms remain in the bioindicator after the sterilization process is completed. However, conventional sterility measurement or detection procedures require at least one full day for direct (and at least 20 minutes for indirect) measurements of microorganism survival. Additionally, most conventional procedures rely on indirect measurement of microorganism survival, (e.g., the accumulation of a substrate from an enzymatic reaction)_and do not quantify the microorganism survival. According to embodiments of the present disclosure, the bioindicators can be used in verification systems and methods that rely on direct measurement of microorganism survival, can return measurement results in a significantly reduced time frame (e.g., in 5 minutes or less), and can quantify the amount of microorganism survival.

[0023] As noted generally above, the genetically modified test microorganism n used in the bioindicator (Bl) is typically selected from those microorganisms that have a known resistance to sterilization or to the particular sterilization process being tested (i.e., those microorganisms that are not easily killed by sterilization or the particular sterilization process being tested). As such, the genetically modified test

microorganism used in the bioindicator is a good indicator of the success or failure of the sterilization process.

[0024] In some embodiments of the present disclosure, the genetically modified test microorganisms are in the form of genetically modified microbial spores. Bacterial endospores, rather than the vegetative form of the bacteria, are very resilient and may remain in their dormant state for years, whereas the vegetative form is more easily inactivated. As a result, a sterilization process that inactivates a bacterial endospore provides a high degree of confidence that all (or substantially all) microorganisms were inactivated during the sterilization process. As used herein, the term "substantially" is used as a term of approximation and not as a term of degree, and is intended to account for inherent inaccuracies (or standard deviations) in the measurement, detection or observation of certain features, parameters or properties. For example, the inactivation of "substantially all microorganisms" means that any microorganisms remaining after the sterilization procedure are undetectable, unquantifiable, or negligible.

[0025] According to embodiments of the present disclosure, the genetically modified test microorganism(s) in the bioindicator are genetically modified to express an exogenous reporter gene that expresses an optically detectable signal. Detection of the optically detectable signal is carried out either by naked eye observation of the bioindicator after sterilization, or in a detection apparatus (or device) into which the bioindicator is placed after being subjected to (or exposed to) the sterilization process. If the detection apparatus (or the naked eye) detects the optically detectable signal, this is indicative of the presence of viable genetically modified test microorganism in the bioindicator, which in turn, indicates that the sterilization process was unsuccessful. If, however, the detection apparatus (or the naked eye) does not detect the optically detectable signal, this is indicative of either no remaining viable genetically modified microorganism or no detectable amount of remaining viable genetically modified microorganism, which in turn, indicates that the sterilization process was successful.

[0026] According to some embodiments, the bioindicator includes a housing in which the genetically modified test microorganism is housed. Figs. 1 and 2 depict exemplary bioindicators Bl according to embodiments of the present invention. As shown in Figs. 1 and 2, a bioindicator according to embodiments of the present disclosure includes a housing H with an interior chamber C. Inside the interior chamber C is housed genetically modified test microorganisms 1 , which are

immobilized between an optically transparent window 3 in the housing and a semipermeable matrix 2 positioned in the interior chamber between the individual genetically modified test microorganisms 1 and the optically transparent window 3.

[0027] As shown in Fig. 1 , in some embodiments, the housing H further includes an aperture (or opening) 4 that allows for delivery of the expression composition (e.g., a germinant and/or growth media mixture) to the semi-permeable matrix 1 via, e.g., a syringe or any other suitable introduction device. The user may introduce the expression composition through the aperture 4 after the bioindicator Bl has been subjected to the sterilization process undergoing sterility assurance testing.

[0028] Alternatively, as shown in Fig. 2, the bioindicator Bl may further include a breakable capsule or pouch 5 housing the expression composition isolated from the individual genetically modified test microorganisms 2. The breakable capsule or pouch 5 serves to maintain the expression composition isolated from the genetically modified test microorganisms 2 during storage of the bioindicator Bl, and before and during the sterilization process undergoing sterility assurance testing. After the bioindicator Bl has been subjected to the sterilization process, the user then causes the breakable capsule or pouch 5 to rupture, thereby causing the expression composition to be released from the breakable capsule or pouch 5 and contact the genetically modified test microorganisms 2 within the interior chamber C of the housing H. The breakable capsule or pouch 5 may be ruptured by any suitable means, including but not limited to the application of a crushing (e.g., either with the user's bare hands or with a crushing tool) or piercing (e.g., with a syringe or other sharp tool) force, or other force sufficient to cause the pouch to rupture. [0029] Any suitable microorganism may be used in the genetically modified test microorganism in the bioindicator so long as the microorganism has at least some resistance to sterilization or the specific sterilization process being tested. Non-limiting examples of suitable microorganisms for use in the genetically modified test microorganism include any bacteria or other organism demonstrating resistance to sterilization, or to the specific sterilization method being tested. Non-limiting examples of suitable bacteria include any of the spore-forming bacteria including, but not limited to Geobacillus stearothermophilus, Bacillus subtillus, Bacillus atropheus, Bacillus cereus, Bacillus anthracis, Clostridium difficile, Clostridium botulinum, Clostridium perfringens, Paenibacillus larvae, and Paenibacillus polymyxa, as well as any other suitable spore-forming bacteria.

[0030] In some embodiments of the present disclosure, the reporter gene 8 is not necessarily a protein-coding gene, but may express any of the fluorescent proteins.

Non-limiting examples of suitable fluorescent reporter proteins include any of the green fluorescent proteins (GFP) derived from Aequorea victoria, including GFP, enhanced GFP (eGFP), yellow fluorescent protein (YFP), and superfolder GFP (sfGFP); any of the red fluorescent proteins derived from Discosoma sp. (Ds), including DsRed and its mutants such as monomeric (m) red fluorescent protein (imRFP), mCherry, and tandem-dimer Tomato (tdTomato); as well as any other fluorescent protein (i.e., a fluorophore) including those derived from the cyanobacterial alpha-phycocyanobilin proteins. Protocols and selection of fluorescent report proteins are known in the art as described, e.g., in "Fluorescent Proteins: A Cell Biologist's User Guide," Trends Cell Biol. 19:649-655, (doi: 10.1016/j.tcb.2009.08.002), the entire contents of which are herein incorporated by reference.

[0031] According to some embodiments of the present disclosure, the genetic modification of the genetically modified test microorganisms 2 may include transformation of a plasmid P (e.g., shown in Figs. 3-6) into the test microorganism (e.g., into the unmodified version of the test microorganism). The plasmid P regulates expression of the reporter gene 8. As shown generally in Fig. 3 (and more specifically in Figs. 4-6), the plasmid P regulates this expression by inclusion of any one or more suitable regulatory elements 9 (shown in Fig. 3). Non-limiting examples of suitable such regulatory elements 9 include constitutive, chemically inducible, physically inducible, or other regulated promoters, as well as enhancers and any transcriptional elements for limiting expression to one or more phases of bacterial germination and outgrowth. With reference to Figs. 3-6, the plasmid P may also contain one or more origins of replication (ORI) 6 suitable for replication of the plasmid in Geobacillus spp. and Bacillus spp. Non-limiting examples of suitable ORIs include pNW33N, repBSTI , and pBC16. In some embodiments of the present disclosure, the introduction of a reporter gene into the spore-forming bacteria may be accomplished by transformation of the bacteria with a selected plasmid P encoding the reporter gene. Bacteria transformed with the plasmid P may be selected by growth in the presence of an antibiotic corresponding to an antibiotic resistance gene (abR) 7 also encoded on the plasmid. Alternatively introduction of a reporter gene into the spore-forming bacteria may be accomplished the integration of the reporter gene and a selection marker (e.g., an antibiotic resistance gene or a disruption of a native bacterial gene) into the bacterial genome.

[0032] In some embodiments of the present disclosure, the genetically modified test microorganism may include genetically modified Geobacillus stearothermophilus bacterium. According to some embodiments, the Geobacillus stearothermophilus bacterium may be genetically modified by means of transformation with a plasmid capable of expressing any thermostable fluorescent protein (TS-FP) reporter. The plasmid may also contain a gene encoding resistance to an antibiotic allowing for selective culture of the genetically modified (i.e., transformed) test microorganisms carrying the plasmid. In some embodiments, a preparation of endospores of this genetically modified bacterium may be used as the genetically modified test

microorganism 2 in the bioindicator Bl. Upon introduction of an expression

composition (e.g., a germinant mixture, for example an aqueous solution of the amino acid L-alanine), and heating of the genetically modified test microorganism within the interior chamber C of the bioindicator, any endospore undergoing the early phases of germination and growth will express TS-FP proteins (as a fluorescent reporter of germination). As discussed above, introduction of the expression composition (e.g., the germinant mixture) may be accomplished by addition of the expression

composition through the aperture 4 in the housing H of the bioindicator Bl, or by rupture of the breakable capsule or pouch 5 including the expression composition to thereby cause the expression composition to contact the genetically modified test microorganisms 2. Additionally, heating of the genetically modified test microorganism within the interior chamber C of the bioindicator may include heating to any suitable germination-permissive temperature, e.g., 50 to 70 °C (degrees centigrade). In some embodiments, the suitable germination-permissive temperature is 55 to 60 °C. In some embodiments, viability of the genetically modified test microorganism (e.g., the endospore) is determined by observation of fluorescence from the location of the genetically modified test microorganisms in the bioindicator. In some embodiments, germination of the endospores is detected by placing the bioindicator (into which the expression composition has been introduced) into a fluorescence detection apparatus, and detecting the fluorescence of the fluorescent reporter proteins using the apparatus. Any suitable fluorimeter or other fluorescence imaging instrument may be used for this purpose. [0033] In some embodiments of the present disclosure, the genetically modified Geobacillus stearothermophilus may be genetically modified by means of

transformation with a plasmid such as the one depicted in Fig. 4. As shown in Fig. 4, the plasmid P incudes a suitable antibiotic resistance gene (abR) (7 in Fig. 3), super- folding GFP (sfGFP) as a thermostable fluorescent protein (i.e., the reporter gene 8 in Fig. 3), and atpA F0F1 ATP synthase subunit alpha promoter (PatpA) as the regulatory element (9 in Fig. 3) for controlling expression of the sfGFP gene. The plasmid shown in Fig. 4 effects expression of the sfGFP reporter upon germination at the same time as expression of the atpA gene. Non-limiting examples of other suitable promoters (i.e., other than the PatpA) for regulation of expression during germination include the promoters for tig, malS, and pupG genes of B. subtilis, and their homologs. Non- limiting examples of suitable antibiotic resistance genes for Geobacillus

stearothermophilus, e.g., providing resistance to antibiotics that are stable at high growth temperatures (e.g., 50 to 70 °C) include the TK101 kanamycin resistance (KanR) gene, and the chloramphenicol acetyltransferase (cam) gene.

[0034] In some embodiments of the present disclosure, the genetically modified Geobacillus stearothermophilus may be genetically modified by means of

transformation with a plasmid such as the one depicted in Fig. 5. As shown in Fig. 5, the plasmid P includes a suitable antibiotic resistance gene (abR) (7 in Fig. 3), and a chemical-inducible promoter (e.g., an IPTG-inducible promoter, for example, Pspac) as the regulatory element 9 for controlling expression of the sfGFP gene (i.e., the reporter gene 9 in Fig. 3). In embodiments using IPTG-inducible promoters (e.g., Pspac), isopropyl β-D-l -thiogalactopyranoside (IPTG) is added to the expression composition (e.g., the germinant mixture) to induce expression of the reporter. While IPTG promoters are disclosed here, it is understood that any other suitable chemically- inducible promoters may be used. For example in some embodiments, xylose- inducible promoters are utilized in the heterologous plasmid. In embodiments using a plasmid with a xylose-inducible promoter, xylose is added to the expression

composition.

[0035] In some embodiments of the present disclosure, the genetically modified Geobacillus stearothermophilus is genetically modified by means of transformation with a plasmid such as the one depicted in Fig. 6. As shown in Fig. 6, the plasmid P includes a suitable antibiotic resistance gene (abR) (7 in Fig. 3), and a constitutive promoter from the lactate dehydrogenase gene, Pldh, as the regulatory element 9 for controlling expression of the sfGFP reporter gene (8 in Fig. 3). In some embodiments, a change in the fluorescence signal detected by an appropriate fluorimeter or fluorescence imaging instrument indicates the production of sfGFP, and thus an increase in metabolic activity.

[0036] In some embodiments of the present disclosure, the genetically modified Geobacillus stearothermophilus is genetically modified by means of integration of a genetic construct encoding a reporter gene including a fluorescent protein fused with a native protein of Geobacillus stearothermophilus capable of expression and

localization of the native protein during sporulation. Accordingly, the detection of fluorescence from these reporter genes serves as an indirect indicator of endospore survival after the sterilization process, which sterilization process causes denaturation, cleavage, or other disruption of the protein structure. In particular, the detection of fluorescence after the sterilization process has completed (i.e., despite the

denaturation, cleavage or other disruption of the protein structure) indicates that the sterilization process did not effectively inactivate the underlying endospore.

Accordingly, detection of the fluorescence indicates that the sterilization process being tested was unsuccessful. [0037] In some embodiments, the genetically modified test microorganism is engineered to express a non-protein-coding reporter gene encoding an RNA aptamer detectable by its luminescent properties upon complexation with an RNA-binding chemical. In some embodiments, the RNA-binding chemical may be provided with the germinant or growth mixture. A suitable RNA-binding molecule includes any RNA- binding molecule that is not detectably fluorescent when not bound to RNA and emits fluorescence when bound to RNA. Non-limiting examples of suitable RNA-binding chemicals that produce luminescence upon binding the encoded RNA aptamer include 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and derivatives of DFHBI including DFHBI-1 T.

[0038] In some embodiments, the genetically engineered test microorganism is engineered to express a bioluminescent reporter protein. Non-limiting examples of bioluminescent reporter proteins include firefly luciferase, aequorin, Renilla luciferase, and related genes.

[0039] According to some embodiments of the present disclosure, systems for sterility assurance (or systems for testing the efficacy of a sterilization process) include a bioindicator (Bl) housing at least one genetically modified test microorganism (e.g., at least one genetically modified endospore). The bioindicator is described herein, and the system may further include a detection apparatus DA for detecting the viability or survival of the genetically modified test microorganism. In some embodiments, as shown in FIGs. 12 and 13, the detection apparatus DA may include one or more sample well S or other sample receptacle for receiving the bioindicator. There is no limit to the number of sample wells or sample receptacles in the detection apparatus, and any given detection apparatus may have any number of sample wells. In practice, however, the number of sample wells S will be dictated by the size of the detection apparatus DA, and therefore the space available for the sample wells S. [0040] The system also includes an expression composition EC. The expression composition may be housed within the bioindicator Bl (for example, within in a breakable pouch) as described above, or may be housed within (see FIG. 12) or otherwise in communication with the detection apparatus DA, such as via connection to an external reservoir (see FIG. 13). The detection apparatus DA may be configured or programmed to pump or otherwise deliver the expression composition over the bioindicators or otherwise to the genetically modified test microorganism within the bioindicator. To that end, in some embodiments, the detection apparatus DA may include an expression composition reservoir R and a pump P in communication with the expression composition reservoir R and the sample wells S. For example, as shown in FIG. 12, the expression composition reservoir R may be housed within the detection apparatus DA. Alternatively, as shown in FIG. 13, the expression

composition reservoir R may be external of the detection apparatus DA, but in communication with a pump P in the detection apparatus which is configured to pump the expression composition from the expression composition reservoir R to the sample wells S or otherwise into the bioindicators Bl within the sample wells S.

[0041] In use, the bioindicator Bl is exposed to the sterilization process being tested. After completion of the sterilization cycle, the expression composition is introduced to the genetically modified test microorganism in the bioindicator Bl. For example, the expression composition may be introduced by hand, e.g., by syringing the expression composition into the bioindicator through an aperture in the bioindicator. In some embodiments, however, the expression composition may be introduced by breaking a breakable pouch housing the expression composition that resides in the bioindicator. Alternatively, as discussed above, the expression composition may be introduced by pumping it from a reservoir (either internal or external to the detection apparatus) to the sample well in the detection apparatus. The detection apparatus is then used to detect the presence or absence of an optically detectable signal from the genetically modified test microorganism. The detection apparatus is configured to return a positive result if the optically detectable signal is detected, which positive result indicates the presence of viable genetically modified test microorganisms, and therefore a failure of the sterilization process. The detection apparatus is also configured to return a negative result if the optically detectable signal is not detected, which negative result indicates either that no viable genetically modified test

microorganism were present, or that none were detected. The negative result, therefore, indicates a successful sterilization process.

[0042] According to some embodiments of the present disclosure, methods for testing the efficacy of a sterilization process include subjecting a bioindicator housing a genetically modified microorganism (e.g., a genetically modified bacterial endospore) to the sterilization process to be tested, and determining the viability or survival of any of the genetically modified test microorganism remaining in the bioindicator after the sterilization process has been completed. The genetically modified test microorganism and the bioindicator are as described herein. According to embodiments of the present disclosure, the bioindicator is placed in the sterilization area (e.g., chamber) or otherwise exposed to the sterilization process to be tested. After the sterilization process, an expression composition is added to or otherwise combined with the genetically modified test organism in order to induce expression of the heterologous reporter gene. The expression composition is as described herein, and may be introduced to the genetically modified test microorganism by any suitable methods or means. For example, in embodiments in which the expression composition is contained in a breakable pouch in the bioindicator (as described herein), the

expression composition may be introduced by breaking the pouch, thereby releasing the expression composition, which will then contact the genetically modified test microorganism in the bioindicator. In some embodiments, however, as also described herein, the expression composition or some of the reagents of the expression composition may be delivered to the genetically modified test microorganism in the bioindicator by, e.g., syringing the expression composition through an aperture in the bioindicator housing. In some embodiments, for example, as discussed above in connection with the system for sterility assurance, the expression composition may be pumped from an expression composition reservoir (either internal or external to the detection apparatus) to the bioindicator and the genetically modified test

microorganism in the bioindicator. Upon introducing or combining the expression composition with the genetically modified test microorganism, any viable

microorganism that survived the sterilization process will express the heterologous reporter gene encoding an optically detectable signal. The optically detectable signal may be observed by the naked eye or it may be measured (e.g., read) using a detection apparatus, such as an optical detector (e.g., a fluorimeter). The positive detection of the optical signal indicates the survival or viability of at least some of the genetically modified test microorganisms, and therefore that the sterilization process was not successful. Conversely, the negative detection of the optical signal (i.e., the optical signal was not detected) indicates either that no genetically modified test microorganisms survived the sterilization process, or that no living genetically modified test microorganisms were detected. In either case, the negative detection indicates that the sterilization process was successful.

[0043] The following Examples are presented for illustrative purposes only, and do not limit the scope or content of the present application.

EXAMPLES [0044] Experimental Design. Plasmid design and construction was carried out using pNW33N as a backbone (Fig. 7) for the gene and using a RPIsl promoter with a Geobacillus optimized RBS.and the coding sequence for fast folder thermal stable yellow fluorescent protein (fftsYFP) as respectively described in Reeve et al., 2016, "The Geobacillus plasmid set: a modular toolkit for thermophile engineering," ACS Synthetic Biology, 5(12), 1342-1347 and Nagai et al., 2002, "A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications," Nature biotechnology, 20(1 ), 87-90, the entire contents of both of which are herein incorporated by reference. pNW33N plasmid was ordered from Bacillus Genetic Stock Center. Gene with RPIsl promoter, RBS and CDS were ordered from SGI-DNA in a pUC02AMP plasmid. Plasmids were grown out in Invitrogen Library Efficiency DH5a chemically Competent Cells.

[0045] Plasmid Preparation. pNW33N plasmid was attained from Bacillus Genetic Stock Center (ECE136) in JM109. pNW33N Plasmid was propagated in Luria Broth with 25ug/ml_ Chloramphenicol. JM109 colony was grown at 25 °C for 17 hours then 37 °C for 3 hours to make glycerol stock. 50% Glycerol Stock was stored at -80 °C for future use. 10Oul of stock was propagated in 5ml_ of Luria Broth at 36 °C for 16 hours. Culture was spun down at 4000 RPM (4C) for 20 minutes then resuspended in resuspension buffer. (Miniprep was performed used GeneJet Plasmid Miniprep

(Invitrogen)). pUC02-Amp (ampicillin resistance gene) with RPIsl promoter and fast folder thermal stable (ffts) YFP coding sequence (CDS) (Fig. 8) was attained from SGI- DNA in 8ul at a concentration of 3665ng. DECP treated water (H20) was used to dilute the stock to a final concentration of 144ng/uL. Stock solution was diluted 1 :28 using diethylpyrocarbonate (DECP) treated H2O. Diluted stock was used to transform Library Efficiency Competent Cells DH5a (Invitrogen). Transformed colonies were selected for using 25mg/mL ampicillin on LB agar plates. Transformed cells were grown in LB and aliquoted out into 50% Glycerol Stocks. Single colonies were picked and grown in 5mL of 25mg/mL amp. Cultures were spun down in a centrifuge at 4000 rpm for 20 minutes then resuspended in buffer. (Miniprep was performed using

GeneJet Plasmid Miniprep. (Invitrogen). pUC02-amp with RPIsl /fftsYFP insert was digested with Notl. Inset size was verify by gel electrophoresis in 1 .5% agarose gel. pNW33N was digested with BamHI. Backbone size was verified by gel electrophoresis in 1 .5% agarose gel. Both the inset and the backbone were blunt ended using Blunt Ending Kit from Invitrogen. The backbone was dephosphorylated for blunt end ligation to occur properly. pNW33N was transfected into DH5a competent cells and grown on 25mg/ml chloramphenicol plates. Colonies were selected to be grown out for miniprep. An aliquot of the culture selected for transformation of Geobacillus was stored in 50% glycerol stock. pNW33N with fftsYFP insert (Fig. 9) was verified by restriction digest and gel electrophoresis.

[0046] Transformation of Geobacillus stearothermophilus.

[0047] Conjugative Plasmid Transfer. Geobacillus stearothermophilus ATCC 7953 cells were grown in 9mL TSB (tryptic soy broth) shaking at 55 °C for 16 hours. E. coli transformed pNW33N-fftsYFP was grown in 5mL TSB at 36 °C for 16 hours. 1 imL of the E. coli culture was added to the Geobacillus stearothermophilus culture and the mixture of organism was incubated at 36C for 30 minutes. Then the culture was immediately placed on ice for 5 minutes. All 9mL were then vacuum filtered onto Millipore Mixed Cellulose Esters Membrane (AABG). The filter was then transfer to LB agar plates with 25mg/mL chloramphenicol and allowed to incubate for 16 hours at 36 °C. The filter was then removed and placed into 5mL TSB and vortex for 5 minutes. 1 imL of the recovered suspension was spread plated onto TSB plates with 5mg/mL chloramphenicol. Single colonies were picked and imaged under Nikon Eclipse 80e using fluorescent microscopy. [0048] Electroporation. Geobacillus stearthermophilus ATCC 7953 was grown overnight in TSB at 55 °C. Transformed E.coli with pNW33N plasmid was grown in 25mg/ml_ Chloramphenicol for 16 hours. Plasmids were isolated using GeneJET™

Miniprep kit from (Invitrogen.) Geobacillus stearothermophilus was transformed using the electroporation protocol described in KananaviciGte et al. 2015, "Geobacillus stearothermophilus NUB3621 R genetic transformation by electroporation," Biologija, 61 (3-4, the entire content of which is herein incorporated by reference. A Bio-Rad Genepulse ShockPod™ was used with 0.4cm electrode gap Gene Pulser® Cuvettes. Transformed Geobacillus was grown in TSB with 5mg/ml_ chloramphenicol for 16 hours and then placed onto 5mg/ml_ chloramphenicol plates.

[0049] Imaging. Colonies from pNW33N-fftsYFP transformed Geobacillus stearothermophilus were placed into 1 ml_ TSB with 5mg/ml_ Chloramphenicol. Images were taken after 90 minutes using fluorescent microscopy to determine whether fluorescence was observed. Figs. 10A and 10B show Geobacillus stearothermophilus transfected with pNW33N-fftsYFP plasmid using conjugative plasmid transfer. Fig. 10A shows fluorescent microscopy using a Semrock brightline LED-YFP-A-NTE filter cube. Image was taken with 500ms exposure using Infinity 3s Lumenera Camera at 40x magnification. Fig. 10B shows phase imaging using 40x phase objective. This imaging shows the transformation of ffts YFP into Geobacillus stearothermophilus.

[0050] Figs. 1 1 A and 1 1 B show images of Geobacillus stearothermophilus transfected with pNW33N-fftsYFP plasmid using electroporation. Fig. 10A shows fluorescent microscopy using a DAPI filter cube; the image was taken with 500ms exposure using Infinity 3s Lumenera Camera at 40x magnification. Fig. 10B shows fluorescent microscopy using Semrock Brightline LED-YFP-A-NTE filter cube; the image was taken with 500ms exposure using Infinity 3s Lumenera Camera at 40x magnification. [0051] While the present invention has been illustrated and described with reference to certain exemplary embodiments, those of ordinary skill in the art will understand that various modifications and changes may be made to the described embodiments without departing from the spirit and scope of the present invention, as defined in the following claims.

Claims

WHAT IS CLAIMED IS:

1 . A method for testing the efficacy of a sterilization process, the method comprising:

subjecting a bioindicator to a sterilization process, the bioindicator comprising a genetically modified test organism modified with a plasmid comprising a heterologous reporter gene capable of producing an optically detectable signal;

after the sterilization process, introducing an expression composition to the genetically modified test organism, the expression composition being capable of inducing expression of the heterologous reporter gene;

detecting the presence or absence of the optically detectable signal; and determining that the sterilization process was successful or unsuccessful based on the presence or absence of the optically detectable signal.

2. The method of claim 1 , wherein the genetically modified test

microorganism is a bacterial endospore.

3. The method of claim 2, wherein the bacterial endospore is selected from Geobacillus stearothermophilus, Bacillus subtilis, Bacillus atrophaeus, Bacillus cereus,

Bacillus anthracis, Clostridium difficile, Clostridium botulinum, Clostridium perfringens, Paenibacillus larvae, or Paenibacillus polymyxa.

4. The method of claim 1 , wherein the optically detectable signal is luminescence, fluorescence, phosphorescence, absorbance, or refractivity.

5. The method of claim 1 , wherein the heterologous reporter gene is a reporter protein or an RNA aptamer.

6. The method of claim 5, wherein the heterologous reporter gene is an RNA aptamer and the composition capable of inducing expression of the heterologous reporter gene further comprises an RNA binding molecule capable of producing luminescence upon binding the RNA aptamer.

7. The method of claim 5, wherein the reporter protein is a fluorescent reporter protein or a bioluminescent reporter protein.

8. The method of claim 1 , wherein the expression composition capable of inducing expression of the heterologous reporter gene comprises L-alanine.

9. The method of claim 1 , wherein the expression composition capable of inducing expression of the heterologous gene comprises isopropyl β-D-l - thiogalactopyranoside (IPTG) or xylose.

10. A system for testing the efficacy of a sterilization process, the system comprising:

a bioindicator comprising a housing having an optically transparent window and an interior chamber;

a genetically modified test microorganism in the interior chamber of the bioindicator, the genetically modified test microorganism being modified with a plasmid comprising a heterologous reporter gene, the heterologous reporter gene being capable of producing an optically detectable signal upon exposure to an expression composition capable of inducing expression of the heterologous reporter gene; and a detection apparatus configured to detect the presence or absence of the optically detectable signal.

1 1 . The system of claim 10, wherein the genetically modified test

microorganism is a bacterial endospore.

12. The system of claim 1 1 , wherein the bacterial endospore is selected from Geobacillus stearothermophilus, Bacillus subtilis, Bacillus atrophaeus, Bacillus cereus, Bacillus anthracis, Clostridium difficile, Clostridium botulinum, Clostridium perfringens, Paenibacillus larvae, or Paenibacillus polymyxa.

13. The system of claim 10, wherein the optically detectable signal is luminescence, fluorescence, phosphorescence, absorbance, or refractivity.

14. The system of claim 10, wherein the heterologous reporter gene is a reporter protein or an RNA aptamer.

15. The system of claim 14, wherein the reporter protein is a fluorescent reporter protein or a bioluminescent reporter protein.

16. The system of claim 10, wherein the heterologous reporter gene is an RNA aptamer and the expression composition comprises an RNA binding molecule capable of producing luminescence upon binding the RNA aptamer.

17. The system of claim 10, wherein the expression composition comprises L-alanine, isopropyl β-D-l -thiogalactopyranoside (IPTG), or xylose.

18. The system of claim 10, wherein the bioindicator further comprises breakable pouch comprising the expression composition.