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WO2020264009A1 - Procédé de fabrication d'indicateurs biologiques - Google Patents

Procédé de fabrication d'indicateurs biologiques Download PDF

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Publication number
WO2020264009A1
WO2020264009A1 PCT/US2020/039401 US2020039401W WO2020264009A1 WO 2020264009 A1 WO2020264009 A1 WO 2020264009A1 US 2020039401 W US2020039401 W US 2020039401W WO 2020264009 A1 WO2020264009 A1 WO 2020264009A1
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WIPO (PCT)
Prior art keywords
spores
superdormant
spore
germinating
labile
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Ceased
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PCT/US2020/039401
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English (en)
Inventor
David Opie
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Noxilizer Inc
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Noxilizer Inc
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Filing date
Publication date
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Publication of WO2020264009A1 publication Critical patent/WO2020264009A1/fr
Anticipated expiration legal-status Critical
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/22—Testing for sterility conditions

Definitions

  • the invention relates to the manufacturing of both biological indicators (Bis) and spore suspensions in which determining the ratio of normally dormant spores to superdormant spores is measured or manipulated.
  • Bis consist of a population of spores on a carrier made of stainless steel, paper, Tyvek or other appropriate material. Bis are used for validating and monitoring processes for disinfection, decontamination and sterilization. Herein, all of these processes will be collectively referred to as sterilization, but it is understood that these methods apply equally to all such processes in which a reduction of microbial contamination is sought.
  • bacterial spores are oftentimes used as Bis to monitor the efficacy of specific sterilization parameters by measuring spore lethality under the given sterilization conditions.
  • Bis allow for the validation of sterilization parameters because if spores are inactivated during a sterilization process, then it is accepted that all microorganisms on a medical product will be inactivated, hence the validation of a sterile medical product (ref. 10). Therefore, it is important to manufacture predictable and reliable Bis to validate different sterilization conditions.
  • spore particles on a BI carrier which includes both normally dormant and superdormant spores.
  • Normally dormant spores become enumerable during common germination processes.
  • spores are applied to the surface of a plate consisting of a nutritive agar media. After which, the spores grow, form colonies and can be counted.
  • superdormant spores do not become enumerable with exposure to nutrients and instead remain dormant and are called superdormant.
  • a problem with existing manufactured Bis is that the number of spore particles is uncertain. If a manufacturer labels a BI carrier as having a certain number of spores, but there are superdormant spores that were not enumerated during germination processes, then an incorrect spore population is produced and the log-linear response of the BI is degraded. Consequently, an incorrectly manufactured BI (containing an excess of superdormant spores) could lead to an irreproducible sterilization process result.
  • sterilization conditions can yield unexpected results due to inconsistency in determining the true number of spore particles on a BI carrier because the sterilization process can trigger superdormant spores to become germinating spores, becoming enumerable, thereby increasing the number of enumerable spores because of the sterilization process (when only a decrease in population is expected).
  • BI performance consistency is critical for successfully monitoring and validating processes that rely on Bis. For example, with one lot used for initial process development and a new lot of Bis for validation, the non-log linear response can cause validation failure due to the rogue Bis.
  • One cause has been identified as clumping of the spores on the BI carrier, and this clumping protects the spores that are covered by other spores. For example, this problem can be amplified when the BI population is determined by counting normally dormant spores and superdormant population is 300 % or more of the normally dormant population. Such a high superdormant population causes increased spore clumping.
  • the population of superdormant spores varies from one spore crop to another.
  • the inventor has found that between 30 % to 50 % of the spore crops screened have a significant population of superdormant spores.
  • the disclosed methods provide a solution to manufacturing Bis and spore suspensions, wherein both normally dormant and superdormant spores are enumerated, allowing for predictable Bis for sterilization monitoring.
  • Another aspect of the disclosed manufacturing methods triggers at least a portion of the superdormant spores to become germinating spores prior to applying these spores to the BI carrier.
  • a problem in the art of manufacturing Bis and spore suspensions is determining the number of spore particles on the BI carrier.
  • the number of spore particles is defined as the number of both normally dormant and superdormant spores.
  • Normally dormant spores are spores that can germinate and are therefore enumerable.
  • superdormant spores must be stimulated, or triggered, to germinate and become enumerable with the common enumeration methods.
  • Triggering mechanisms are often additionally necessary to determine the number of spore particles because triggering mechanisms allow for the conversion of superdormant spores to germinating spores, and in turn renders superdormant spores enumerable.
  • the triggering mechanisms include heat-shocking, acid shocking, chemical exposure or other means.
  • the triggering mechanism permits the heat labile, acid labile, protein labile to be counted. It is important to note that a portion of the superdormant spore population may convert to germinating spores during one triggering mechanism and a different cohort of superdormant spores can convert to germinating spores in response to a different triggering mechanism. Thus, it is useful to perform more than one triggering step to gain a full understanding of the triggerable spore particle population.
  • a further aspect of this invention is the order of performing the triggering mechanism.
  • Heat-shocking spores is a common microbiological practice and the method involves exposing a spore suspension to heat such as a water bath of 95°C or warmer for a pre-determined amount of time (e.g., 10 minutes). Heat-shocking spores can convert heat-labile superdormant spores to germinating spores. An estimate of the superdormant population in this case is the difference between the number of heat-labile spores and the number of normally dormant spores.
  • spore suspensions and Bis with known normally dormant and superdormant populations can be achieved by performing the three triggering mechanisms.
  • An alternative to using triggering mechanisms is a method of counting all spore particles (normally dormant and superdormant) and comparing this number to the number of normally dormant, enumerated spores. For example, subtracting the number of normally dormant spores from the number of counted spore particles yields the total number of superdormant spores.
  • Such counting methods can be flow cytometry, image cytometry, or other particle counting means.
  • the cytometric evaluation of a spore suspensions in Table 1 shows the total number of spore particles divided by the non-heat shock population of spores.
  • Spore suspension 1 shows 136% more spores measured by image cytometry versus enumeration. While spore suspension 2 shows fewer spores found with image cytometry, this is likely just variation in the preparation of the samples. Spore suspension 3 and 4 have 166% and 451% more spores as measured with image cytometry. This is a typical distribution of normally dormant and superdormant spores.
  • Table 1 Selected spore suspensions from different spore crops showing the ratio of cytometric population to the non-heat shock population.
  • Fig.l A comparison of spore population recovered from Bis using heat shock, acid shock and chemical treatment. In this case, both acid shocking and chemical treatment (urea) triggered superdormant spores to become normally germinating.
  • Fig. 2 The observation of population increase during a sterilization process that uses N02 as a component of the sterilization process.
  • Biological indicators that are used for monitoring sterilization and biodecontamination processes consist of spores applied to the surface of a carrier.
  • the spores can be any organism that is appropriate for use as the indicating organism for a sterilant exposure process and the population of spores per carrier is usually 10,000 (104) to more than 1,000,000 (106) spores.
  • the carrier may be made of stainless steel, paper, Tyvek, glass, quartz filter media, and the like. Often, these Bis are packaged in simple Tyvek pouches and in some cases the Bis are built into a more convenient, self-contained biological indicator package or used in a process challenge device.
  • All spores are dormant forms of organism. After germination, normally dormant spores become metabolizing and replicating organisms, referred to as vegetative organisms.
  • the trigger for germination may be the presence of nutrients or other chemicals in the recovery and nutritive media.
  • the method of testing viability of a spores on a BI is to use a recovery process.
  • This recovery process can be either to test for any viable spores on a BI, or the recovery process may be to enumerate, or count the viable spores on a BI carrier.
  • a non-quantitative test for any viable spores on a BI carrier consists of placing the entire BI in liquid growth medium, and after a period of time, evaluating the liquid growth medium for evidence of spores becoming vegetative organisms, shown by turbidity or the opaqueness of the liquid growth media.
  • a quantitative method is an enumeration recovery method which counts the number of viable spores on a BI, using serial dilution and plating procedures. These are common microbiological techniques to those familiar with the testing Bis.
  • the method is applied to samples selected from a manufacturer batch to determine whether the batch meets production quality metrics and/or to characterize the batch so that it may be sold with a correct labeling.
  • the process may be used to validate a batch of Bis received by a customer who would like to be certain that the labeled spore population is correct.
  • the process may be applied to trigger spores prior to applying a suspension of spores to a carrier to prepare the Bis.
  • a sterilization process consists of exposing the load to be sterilized to the sterilizing agent (e.g., steam, sterilizing gas, etc.) for a specified time. After exposure to the sterilant exposure process, the spores can be recovered and evaluated for spore viability.
  • the rate at which exposed spores are inactivated (killed) will follow a log-linear response, where one log of the spore population is inactivated during each increment of the sterilant exposure process. Therefore, the time and exposure conditions required for the inactivation of the known population of spores is predictable and defines the resistance of the spores to the sterilant exposure process.
  • the layering of the spores is a critical factor that is not so easily manipulated. Furthermore, the degree of layering will increase as the number of spores (normally dormant and superdormant) on the carrier increases.
  • the spores are typically applied to the BI carrier as a liquid spore suspension, the volume of the liquid inoculum, the conditions for drying the liquid spore suspension, and other factors can affect the layering of the spores. However, even with these factors controlled, spore layering on the BI carrier is amplified by the presence of superdormant spores.
  • Another factor that contributes to layering is the total population of the spores on the carrier.
  • the total number of spore population on a carrier is known to change the apparent BI resistance, due to increasing the number of spores on the carrier increases the amount of layering may occur as shown in Table 2. (ref. 9). Applying more spores to the carrier surface than are needed to achieve the target population is detrimental to consistent and reproducible BI performance.
  • Table 2 shows the response of Geobacillus stearothermophilus Bis with varying spore populations and exposed to increasing chlorine dioxide sterilant gas (mg-hr/L) at 65% RH to 75% RH.
  • Superdormant spores can be converted to germinating spores when exposed to triggering mechanism such as heat shock, acid shock, or a chemical treatment.
  • triggering mechanism such as heat shock, acid shock, or a chemical treatment.
  • chemical for treatment include urea, cationic surfactants and proteins that can bind or react to germination binding sites on the spore.
  • Fig. 1 shows an example of a spore suspension where 2 x 106 spores present germinate with the normal enumeration recovery techniques (the 0 minutes exposure time to 0.1 M HC1 datum on the graph), and where these recovery techniques included heat-shocking the spores.
  • the normal enumeration recovery techniques included heat-shocking the spores.
  • the enumerated population exhibits a 400 % increase in the number of spores counted after 10 minutes of 0.1 M HC1 exposure.
  • Fig. 1 shows that the common microbiological technique of heat- shocking spores is not sufficient in accounting for the total amount of spore particles.
  • Heat- shocking the spores allowed for the conversion of normally dormant spores, but with twenty minutes of heat exposure at >95 °C, 2 x 106 spores were recovered, compared to the almost 5 x 106 spores recovered after 20 minutes of exposure to 0.1 M HC1, proving that the heat-shocking method is not sufficient in determining the spore particle population and additional triggering mechanisms are required.
  • the suspension may need to be washed to neutralize triggering treatment and the remove unwanted residuals of the triggering treatment. This is done by diluting the spore suspension, centrifuging the diluted spore suspension, removing the supernatant, and resuspending the spores in suspension. The washing may need to be repeated to result in a clean spore suspension.
  • Another embodiment of this invention is the order of the triggering steps. For example, the inventor found that performing acid shocking of the spore suspension prior to heat shocking of the spore suspension results in Bis with the greatest germinating spore population and the fewest superdormant spores.
  • the term“excess” relates to BI carriers that do not have a superdormant population exceeding 30% of the total amount of spore particles. This allows for controlling the spore population on BI indicators and any BI carriers that have an excess of superdormant spores will not be used for manufacturing purposes.
  • This disclosure also includes methods of manufacturing spore suspension for Bis in which the population of superdormant spores is determined.
  • spore suspension and Bis are not tested to determine the fraction of superdormant spores.
  • the BI manufacturer can either choose to not use that spore suspension, or the superdormant spores can be converted to normally geminating spores using heat, acid, or protein or treatments.
  • embodiments include determining the superdormant spore population by spore particle counting via cytometric methods such as flow cytometry or image cytometry and comparing the particle count with the enumeration recovery results. This might include using at least two techniques of counting the spores applied to a biological indicator carrier and using the difference from counting techniques to characterize the quality of the biological indicator.
  • Identifying the presence of a significant fraction of super dormant spores can be used as one criterion for screening spore crops that might be used for manufacturing Bis.
  • triggering can be used for converting superdormant spores to germinating spores prior to applying the spores to the BI carrier.
  • Embodiments include the following examples:
  • a method of manufacturing a biological indicator (BI) comprising: enumerating spores with at least two different methods, comprising: germinating untreated spores to determine a quantity of normally dormant spores on the BI; and performing a triggering mechanism to determine the presence of superdormant spores on the BI.
  • BI biological indicator
  • [071] 28 The method of example 18 wherein a protein exposure is used as a trigger to convert superdormant spores to germinating spores and following the protein exposure, the spores are enumerated using germination of the protein-exposed spores by enumerating growing colonies of the BI organism to determine the amount of protein labile superdormant spores.
  • [072] 29 The method of example 28 wherein at least a portion of the superdormant population is estimated as the difference between the number of protein-exposed spores and the normally dormant spores.
  • [073] 30 A method of manufacturing a spore suspension used for a biological indicator (BI) wherein spore particles are enumerated with at least two distinct methods, wherein one method involves germination of untreated spores to determine the quantity of normally dormant spores on the BI and the second method involves performing a triggering mechanism to determine the presence of superdormant spores on the BI.
  • BI biological indicator
  • the method of example 30 wherein the number of normally dormant spores is determined by the germination method, which includes enumerating growing colonies of the BI organism, wherein growing colonies are a group of reproducing organisms that when diluted arise from one germinating spore.
  • BI biological indicator
  • a method of manufacturing biological indicator (BI) wherein triggering spores is performed on a spore suspension prior to applying the spores to the BI carrier.
  • the method of example 62 includes removing supernatant from the centrifuged and stratified solution.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
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  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés se rapportant à la fabrication d'indicateurs biologiques et de suspensions de spores, les particules de spores étant énumérées à l'aide d'au moins un procédé parmi plusieurs procédés. Lesdits procédés comprennent la cytométrie de flux, la cytométrie d'image, des traitements de germination et des mécanismes de déclenchement tels que le choc thermique, le choc acide ou des expositions chimiques, afin de déterminer le rapport des spores normalement dormantes aux spores super-dormantes. Avant d'appliquer les spores au support d'indicateur biologique pour la fabrication, les spores peuvent subir l'un des mécanismes de déclenchement décrits. De cette manière, les populations de spores de l'indicateur biologique peuvent être régulées au moyen de la fabrication d'indicateurs biologiques dans lesquels moins de 30 % de la population de particules de spores comprend des spores super-dormantes.
PCT/US2020/039401 2019-06-24 2020-06-24 Procédé de fabrication d'indicateurs biologiques Ceased WO2020264009A1 (fr)

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US201962865361P 2019-06-24 2019-06-24
US62/865,361 2019-06-24

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100166603A1 (en) * 2008-10-31 2010-07-01 Noxilizer, Inc. Powder Sterilization
US20120315622A1 (en) * 2011-05-09 2012-12-13 Rotman M Boris System for detecting and enumerating biological particles
US20140220662A1 (en) * 2013-02-06 2014-08-07 Envera, Llc Dried spore germinative compound mixtures
WO2015126251A1 (fr) * 2014-02-20 2015-08-27 Stichting Top Institute Food And Nutrition Micro-organismes thermorésistants
US20170265463A1 (en) * 2016-03-15 2017-09-21 Case Western Reserve University Sporicidal composition
US20190098915A1 (en) * 2017-10-04 2019-04-04 NCH Life Sciences LLC Nutrient-spore formulations and uses thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100166603A1 (en) * 2008-10-31 2010-07-01 Noxilizer, Inc. Powder Sterilization
US20120315622A1 (en) * 2011-05-09 2012-12-13 Rotman M Boris System for detecting and enumerating biological particles
US20140220662A1 (en) * 2013-02-06 2014-08-07 Envera, Llc Dried spore germinative compound mixtures
WO2015126251A1 (fr) * 2014-02-20 2015-08-27 Stichting Top Institute Food And Nutrition Micro-organismes thermorésistants
US20170265463A1 (en) * 2016-03-15 2017-09-21 Case Western Reserve University Sporicidal composition
US20190098915A1 (en) * 2017-10-04 2019-04-04 NCH Life Sciences LLC Nutrient-spore formulations and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SONALI GHOSH, PETER SETLOW: "Isolation and Characterization of Superdormant Spores of Bacillus Species", JOURNAL OF BACTERIOLOGY, vol. 191, no. 6, 26 February 2009 (2009-02-26), pages 1787 - 1797, XP055780160, ISSN: 0021-9193, DOI: 10.1128/JB.01668-08 *
ZHANG YIFAN, MATHYS ALEXANDER: "Superdormant Spores as a Hurdle for Gentle Germination-Inactivation Based Spore Control Strategies", FRONTIERS IN MICROBIOLOGY, vol. 9, 3163, 4 January 2019 (2019-01-04), pages 1 - 10, XP055780161, DOI: 10.3389/fmicb.2018.03163 *

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