WO2023202846A1 - Procédé, système et instrument de mesure pour déterminer la présence de bactéries vivantes dans un échantillon - Google Patents

Procédé, système et instrument de mesure pour déterminer la présence de bactéries vivantes dans un échantillon Download PDF

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Publication number
WO2023202846A1
WO2023202846A1 PCT/EP2023/057905 EP2023057905W WO2023202846A1 WO 2023202846 A1 WO2023202846 A1 WO 2023202846A1 EP 2023057905 W EP2023057905 W EP 2023057905W WO 2023202846 A1 WO2023202846 A1 WO 2023202846A1
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WO
WIPO (PCT)
Prior art keywords
isotope
gas
sample
measuring device
bacteria
Prior art date
Application number
PCT/EP2023/057905
Other languages
German (de)
English (en)
Inventor
Benjamin Scherer
Timo Hillebrand
Elmara Graser
Robert MÖLLER
Thomas Moore
Christiane Ehrling
Florian Krogmann
Original Assignee
Innovative Sensor Technology Ist Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innovative Sensor Technology Ist Ag filed Critical Innovative Sensor Technology Ist Ag
Publication of WO2023202846A1 publication Critical patent/WO2023202846A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath
    • G01N33/4977—Metabolic gas from microbes, cell cultures or plant tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/18—Water
    • G01N33/186—Water using one or more living organisms, e.g. a fish
    • G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms

Definitions

  • the invention relates to a method for determining the presence of living bacteria in a sample using a measuring device, a system and a measuring device.
  • bacteria can form biofilms together with other microorganisms such as algae and fungi that can accumulate in process systems.
  • Hygiene and sterility are essential, particularly in the production of the food and pharmaceutical industries, so that a gap-free design is generally required for all process-contacting sections of measuring devices or other components of the process system in order to make it more difficult for biofilms to accumulate.
  • Bacteria are also found, for example, in the wastewater of sewage treatment plants or can accumulate in cooling towers, so that regular monitoring of the bacteria in the systems is necessary in such plants.
  • ATP adenosine triphosphate is an energy source that occurs in all cells.
  • the ATP can be quantified using optical methods.
  • the ATP test is generally used to check whether there is contamination by microorganisms, such as bacteria or algae.
  • pollutants include, among others:
  • the object underlying the present invention is therefore to provide a method, a measuring device and a system by means of which the presence of bacteria can be determined in a simple manner.
  • the object is achieved by the method according to the invention according to claim 1, the measuring device according to the invention according to claim 8 and the system according to the invention according to claim 13.
  • Measuring device has at least one gas cell, a detection unit and an evaluation unit, the gas cell having a membrane which is permeable to at least one gas, the gas being characteristic of bacteria present in the sample, the method providing at least the following steps:
  • the substrate can be metabolized by bacteria and has at least a first isotope
  • incubating the sample at a predetermined temperature for a predetermined period of time wherein the at least one substrate is at least partially converted into the gas by the bacteria which diffuses into the gas cell, determining an isotope ratio of the first isotope to a second isotope in the gas by means of the detection unit, the first isotope and the second isotope being isotopes of the same chemical element
  • the at least one substrate can be metabolized or converted into a gas by living bacteria, so that the presence of living bacteria can be determined based on an analysis of the gas. Dead bacteria are not detected because they do not metabolize the at least one substrate.
  • the gas may not only be produced by the metabolism of the at least one substrate by the bacteria, but can in principle also come from other sources or be part of the ambient atmosphere. It is possible, for example, that in addition to the at least first substrate added, further substrates are present in the sample, which can be metabolized by the bacteria to form the gas.
  • the gas that diffuses through the membrane into the gas cell can therefore comprise gas that has been metabolized from the at least first substrate and gas from other sources.
  • the at least one substrate has at least one first isotope, which is found in the gas after metabolism by the bacteria.
  • the at least first isotope thus indicates at least part of the gas that was generated by the metabolism of the at least one substrate.
  • the at least one substrate can also have a second isotope of the same chemical element in addition to the at least first isotope, so that the gas generated by the metabolism of the at least one substrate has both the first and the second isotope.
  • the at least one substrate has at least one first isotope or is marked with at least one first isotope.
  • the first isotope selected is an isotope from among the isotopes of the same chemical element that occurs less frequently than at least one other isotope.
  • the isotope labeling method describes the systematic insertion of defined isotopes into organic molecules and is particularly known from organic chemistry, where the method is used to break down reaction mechanisms.
  • microorganisms such as bacteria.
  • the analysis of microorganisms is often carried out semi-quantitatively by comparison with a reference value, as in the method according to the invention.
  • a precise determination of the concentration is not possible, among other reasons, because it may be that some of the living bacteria are inactive during the incubation of the sample and do not metabolize the at least one substrate, although in principle they are able to do so.
  • the process steps of presenting the sample, adding the at least one substrate and incubating the sample can be carried out in a device designed for this purpose.
  • the measuring device can, for example, include a measuring cell.
  • a container can be provided which is designed to carry out the mentioned method steps.
  • a substrate which is suitable for the metabolism of a defined bacterial species is added as substrate.
  • a substrate that can be metabolized by a defined bacterial species it is possible to determine the presence of the defined bacterial species and to achieve selectivity with regard to the defined bacterial species.
  • a sugar is added as a substrate.
  • Adding sugar is suitable for Escherichia coli, for example.
  • sugar for example in the form of glucose
  • inulin is a suitable substrate for detecting lactobacilli
  • urea is a suitable substrate for Helicobacter pylori
  • polyurethane is a suitable substrate for Pseudomonas.
  • the sample is incubated with the exclusion of oxygen or with the addition of oxygen. If there are aerobic bacteria in the sample, they need it Oxygen for their metabolism. However, if oxygen is excluded during the incubation of the sample, primarily anaerobic bacteria will metabolize the substrate.
  • a carbon dioxide gas and/or a methane gas is converted as the gas. If the at least one substrate is sugar or urea, for example, it is usually metabolized by bacteria to carbon dioxide gas.
  • a further embodiment provides that an isotope ratio of the carbon isotope 13 C to the carbon isotope 12 C is determined as the isotope ratio.
  • the at least one substrate is marked with the carbon isotope 13 C as at least the first isotope.
  • the more common carbon isotope 12 C can also occur in the at least one substrate if not each of the C atoms of the at least one substrate has been replaced by a 13 C isotope.
  • a temperature of 30°C to 40°C, in particular 37°C, is specified as the predetermined temperature.
  • a time period between 20 and 40 minutes, in particular 30 minutes, is specified as the predetermined time period.
  • At least one type of bacteria is added to the sample in a defined concentration before the sample is incubated, the isotope ratio being compared with a second reference value, it being determined that at least one pollutant is present in the sample, which contains at least one type of bacteria and /or the metabolism of the at least one bacterial species is impaired if the isotope ratio falls below the second reference value.
  • the at least one pollutant is, for example, an antibiotic or a heavy metal.
  • the at least one pollutant impairs, in particular inhibits, slows down or kills, the at least one bacterial species and/or the metabolism of the at least one bacterial species.
  • a specific metabolic activity is determined in the absence of pollutants, which leads to a specific isotope ratio.
  • the metabolic activity decreases, which changes the isotope ratio. If the isotope ratio is smaller than the second reference value, the presence of at least one pollutant in the sample is determined.
  • the object on which the present invention is based is achieved by a measuring device for determining the presence of living bacteria in a sample, the measuring device having at least one gas cell, a measuring cell, a detection unit and an evaluation unit, wherein the measuring cell is designed to receive the sample and at least one substrate, which is metabolizable by bacteria and has at least a first isotope, and to incubate it at a predetermined temperature for a predetermined period of time, the gas cell being arranged adjacent to the measuring cell and a membrane which is permeable to at least one gas, the gas being characteristic of bacteria present in the sample, the detection unit being designed to determine an isotope ratio of the first isotope to a second isotope of the gas, the first isotope and the second isotope are isotopes of the same chemical element, and wherein the evaluation unit is designed to determine the presence of living bacteria based on a comparison of the isotope ratio with a first reference value.
  • the presence of living bacteria in a sample can be easily determined using the measuring device according to the invention.
  • the sample is mixed with at least one substrate in the measuring cell and incubated. During the incubation of the sample, the at least one substrate is at least partially converted into a gas if live bacteria are present in the sample.
  • the gas can also come from other sources, e.g. from the ambient atmosphere that entered the measuring cell with the sample.
  • the at least one substrate is marked with at least a first isotope, so that the at least first isotope is found in the gas after metabolism by living bacteria.
  • the isotope ratio between the first isotope and the second isotope, determined by the detection unit can finally be determined using the computing unit to determine whether living bacteria are present.
  • the gas cell is arranged such that it surrounds the measuring cell.
  • the gas passes particularly easily from the measuring cell into the gas cell.
  • no additional means are necessary for guiding the gas into the gas cell apart from the membrane.
  • the measuring device has a connection area by means of which the measuring device can be connected to a process system. Due to the spatial proximity of the measuring device to the process system, the sample can be quickly transferred from the process system to the measuring device.
  • the measuring device has at least one inlet means, which is designed to introduce the sample from the process system into the measuring device.
  • the inlet means is, for example, a valve. Using the inlet means, a sample from the process system can be introduced into the measuring device at regular intervals and examined for living bacteria using the measuring device.
  • the detection unit is designed to determine the isotope ratio based on an absorption of light irradiated into the gas.
  • the detection unit is a Raman spectrometer.
  • the object on which the present invention is based is achieved by a system with a measuring device and a container for determining the presence of living bacteria in a sample, the measuring device having at least one gas cell, a detection unit and an evaluation unit, the measuring device having at least can be partially introduced into the container, the container being designed to accommodate the sample and at least one substrate, which is metabolizable by bacteria and has at least a first isotope, and to incubate it at a predetermined temperature for a predetermined period of time, the gas cell being a Has a membrane which is permeable to at least one gas, the gas being characteristic of bacteria present in the sample, the detection unit being designed to determine an isotope ratio of the first isotope to a second isotope of the gas, the first isotope and the second isotope is isotopes of the same chemical element, and wherein the evaluation unit is designed to determine the presence of living bacteria based on a comparison of the isotope ratio with a first
  • the sample is present within the container.
  • the container is a tank in which wastewater is treated.
  • the measuring device is at least partially inserted into the container.
  • the measuring device is preferably arranged above the sample.
  • the sample is incubated in the container after the addition of the at least one substrate, the at least one substrate being converted into the gas so that the gas has the at least one isotope.
  • the gas rises through the sample and passes through the membrane into the gas cell of the measuring device.
  • an isotope ratio between the first isotope and the second isotope is determined and the isotope ratio is then compared with a first reference value in order to determine the presence of living bacteria in the sample.
  • the measuring device has a pump which is designed to pump the gas from the gas cell to the detection unit. By pumping the gas towards the detection unit, a larger volume of gas reaches the detection unit, enabling a more precise determination of the isotope ratio.
  • the detection unit is designed to determine the isotope ratio based on an absorption of light irradiated into the gas.
  • the detection unit is a Raman spectrometer.
  • Fig. 1 a first embodiment of a measuring device according to the invention.
  • Fig. 2 a second embodiment of a measuring device according to the invention.
  • Fig. 3 a schematic representation of a system according to the invention.
  • Fig. 1 shows a first embodiment of a measuring device 2 according to the invention for determining the presence of living bacteria in a sample 1.
  • the measuring device 2 comprises a measuring cell 8, which is designed to measure the sample 1 and at least one substrate 7, which can be metabolized by bacteria and has at least one first isotope and incubated at a predetermined temperature for a predetermined period of time.
  • the measuring cell 8 can have, for example, a heating element and possibly a temperature sensor (not shown).
  • the gas cell 3 is arranged adjacent to the measuring cell 8 and has a membrane 6.
  • the gas cell 3 and the measuring cell 8 are at least partially separated by the membrane 6.
  • the gas cell 3 is arranged such that the gas cell 3 surrounds the measuring cell 8.
  • the membrane 6 is permeable to at least one gas which is characteristic of bacteria present in the sample.
  • the measuring device 2 further has a detection unit 4, which is designed to determine an isotope ratio of the first isotope to a second isotope of the gas, the first isotope and the second isotope being isotopes of the same chemical element.
  • the detection unit 4 is designed to determine the isotope ratio based on an absorption of light irradiated into the gas.
  • the detection unit 4 is, for example, a Raman spectrometer.
  • the measuring device 2 includes an evaluation unit 5, which is designed to determine the presence of living bacteria based on a comparison of the isotope ratio with a first reference value.
  • the detection unit 4 and the evaluation unit 5 are, for example, arranged within the gas cell 3.
  • the detection unit 4 and/or the evaluation unit 5 can also be arranged in a different way, for example adjacent to the gas cell 3 or outside the gas cell 3.
  • the measuring device 2 according to the invention from FIG. 1 is suitable for carrying out the method according to the invention.
  • the sample 1 is placed in the measuring cell 8 and then at least one substrate 7 is added to the sample 1.
  • the at least one substrate 7 can be metabolized by bacteria and has at least a first isotope.
  • the sample 1 is shown together with the substrate 7 that has already been added.
  • the substrate can be chosen so that it is suitable for the metabolism of a defined bacterial species.
  • the substrate is sugar.
  • sample 1 is incubated in the measuring cell at a specified temperature for a specified period of time.
  • sample 1 is incubated with the exclusion of oxygen or with the addition of oxygen.
  • Living bacteria at least partially convert the at least one substrate 7 into the gas, which then diffuses from the measuring cell 8 through the membrane 6 into the gas cell 3.
  • the converted gas is, for example, a carbon dioxide gas and/or a methane gas.
  • an isotope ratio of the first isotope to a second isotope in the gas is determined, in particular an isotope ratio of the carbon isotope 13 C to the carbon isotope 12 C.
  • the isotope ratio is then compared with a first reference value using the evaluation unit 5. If the isotope ratio exceeds the first reference value, the evaluation unit 5 determines that living bacteria are present.
  • At least one type of bacteria can be added to sample 1 in a defined concentration before incubating sample 1.
  • the isotope ratio determined by the detection unit 4 is then compared with a second reference value. If the isotope ratio is smaller than the second reference value, it is determined that at least one pollutant is present in sample 1, which affects at least the at least one bacterial species and/or the metabolism of the at least one bacterial species.
  • Fig. 2 shows a second embodiment of the measuring device 2 according to the invention.
  • the components already shown in FIG. 1, such as the measuring cell 8, the gas cell 3 with the membrane 6, the detection unit 4 and the evaluation unit 5, are not shown for the sake of clarity.
  • the measuring device 2 has an optional connection area 9, by means of which the measuring device can be connected to a process system 10.
  • a container 14 is shown as a section of a process system 10.
  • the process system 10 may include other or additional components such as pipes, valves, and pumps.
  • the container 14 contains sample 1, for example.
  • the measuring device can have at least one inlet means 11.
  • a sample 1 from the container can be introduced into the measuring device 2 at regular intervals and in Measuring device 2, the method according to the invention can be carried out.
  • sample 1 can be removed from measuring device 2.
  • the measuring device 2 has an outlet means which is designed to remove the sample 1 from the measuring device 2.
  • a further sample 1 can then be introduced into the measuring device 3 using the inlet means 11. In this way, regular on-site monitoring of the presence of live bacteria in the sample can be carried out.
  • Fig. 3 shows a schematic representation of the system 15 according to the invention with a measuring device 2 and a container 12.
  • the container 12 can be, for example, a container, a tank or a pipe.
  • the measuring device 2 has at least one gas cell 3, a detection unit 4 and an evaluation unit 5.
  • the configurations of these three units have already been described for the measuring device 2 according to the invention in FIG. 1 and apply in an analogous manner to the measuring device 2 of the system 15.
  • the system 15 does not have a measuring cell.
  • the system 15 has the container 12, which is designed to hold the sample 1 and the at least one substrate 7, which is metabolizable by bacteria and has at least a first isotope, and to incubate it at a predetermined temperature for a predetermined period of time.
  • the measuring device 2 can be at least partially inserted into the container 12.
  • the gas cell 3 with the membrane 6 can partially protrude into the container.
  • the gas cell 3 projects in particular into the sample 1 and is at least partially, in particular completely, covered by the sample 1.
  • the detection unit 4 and the evaluation unit 5 are arranged outside the container 12, for example.
  • the measuring device 2 has a pump which is designed to pump the gas from the gas cell 3 to the detection unit 4.
  • the system 15 according to the invention is also suitable for carrying out the method according to the invention.

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  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
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  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédé, système et instrument de mesure permettant de déterminer la présence de bactéries vivantes dans un échantillon (1). Le procédé comprend au moins les étapes suivantes : - chargement initial de l'échantillon (1) ; - ajout d'au moins un substrat (7) à l'échantillon (1), le substrat (7) pouvant être métabolisé par des bactéries et comprenant au moins un premier isotope ; - incubation de l'échantillon (1) à une température prédéterminée pendant une durée prédéterminée, les bactéries transformant au moins partiellement le substrat (7) en gaz se diffusant dans la cellule à gaz (3) ; - détermination d'un rapport isotopique entre le premier isotope et un deuxième isotope dans le gaz à l'aide de l'unité de détection (4), le premier isotope et le deuxième isotope étant des isotopes du même élément chimique ; - comparaison du rapport isotopique avec une première valeur de référence ; et - détermination d'une présence de bactéries vivantes à l'aide de l'unité d'évaluation (5) lorsque le rapport isotopique est supérieur à la première valeur de référence.
PCT/EP2023/057905 2022-04-22 2023-03-28 Procédé, système et instrument de mesure pour déterminer la présence de bactéries vivantes dans un échantillon WO2023202846A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102022109757.9A DE102022109757A1 (de) 2022-04-22 2022-04-22 Verfahren, System und Messgerät zum Ermitteln eines Vorhandenseins von lebenden Bakterien in einer Probe
DE102022109757.9 2022-04-22

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WO2023202846A1 true WO2023202846A1 (fr) 2023-10-26

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011005600A1 (de) * 2011-03-16 2012-09-20 Siemens Aktiengesellschaft Detektion von Organismen und Bestimmung der Resistenz mit Hilfe der Massenspektrometrie
US20130337492A1 (en) * 2012-06-17 2013-12-19 Physical Logic Ag Method and system for real-time, non-invasive monitoring of a biological material in a sealed container
DE102016116767A1 (de) * 2016-09-07 2018-03-08 Rainer Meckenstock Verfahren zur Bestimmung der Biodegradation von organischen Substanzen zu Kohlenstoffdioxid

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140179809A1 (en) 2012-12-12 2014-06-26 Avisa Pharma Inc. Determination Of Location Of Bacterial Load In The Lungs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011005600A1 (de) * 2011-03-16 2012-09-20 Siemens Aktiengesellschaft Detektion von Organismen und Bestimmung der Resistenz mit Hilfe der Massenspektrometrie
US20130337492A1 (en) * 2012-06-17 2013-12-19 Physical Logic Ag Method and system for real-time, non-invasive monitoring of a biological material in a sealed container
DE102016116767A1 (de) * 2016-09-07 2018-03-08 Rainer Meckenstock Verfahren zur Bestimmung der Biodegradation von organischen Substanzen zu Kohlenstoffdioxid

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