WO2015078884A1 - Method and microfluidic assembly for antibiotic susceptibility testing - Google Patents

Method and microfluidic assembly for antibiotic susceptibility testing Download PDF

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Publication number
WO2015078884A1
WO2015078884A1 PCT/EP2014/075602 EP2014075602W WO2015078884A1 WO 2015078884 A1 WO2015078884 A1 WO 2015078884A1 EP 2014075602 W EP2014075602 W EP 2014075602W WO 2015078884 A1 WO2015078884 A1 WO 2015078884A1
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wells
plate
microorganism suspension
susceptibility
antibiotic
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PCT/EP2014/075602
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French (fr)
Inventor
Vadim GOVORUN
Elena ILINA
Alexei PODOPLELOV
Georges ABOU JAOUDÉ
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Alleati Ag
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Publication of WO2015078884A1 publication Critical patent/WO2015078884A1/en

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    • 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
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50853—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates with covers or lids
    • 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
    • C12Q1/18—Testing for antimicrobial activity of a material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates

Definitions

  • the present invention relates to a method and assembly for simultaneous susceptibility testing of microorganisms for different antibiotics and/or chemical agents.
  • the global increase in resistance of bacteria to antimicrobial drugs is generally recognized to be a growing threat for public health.
  • MIC minimum inhibitory concentration
  • MIB minimum bactericidal concentration
  • AST Antibiotic susceptibility testing
  • AST Antibiotic susceptibility testing
  • Small wafers containing antibiotics are placed onto a plate upon which bacteria are growing. If the bacteria are sensitive to the antibiotic, a clear ring, or zone of inhibition, is seen around the wafer indicating poor growth.
  • the inhibition zone diameter can then be correlated to the MIC by taking into account the diffusion rate of the antibiotic through the gel, or by calibrating the assay with susceptibility data measured by, e.g., agar and broth dilution methods. While the Kirby-Bauer method is powerful and versatile, it is a very time consuming process.
  • the method requires several rounds of culturing and incubation, leading to typical test times of 1-3 days depending on the microorganism. This is incompatible with the need for rapid diagnostics and often forces medical practitioners to use initial broad spectrum antibiotic treatment, which is costly, inefficient and increases the problems of antibiotic-resistant bacterial strains in hospital environments. For some infections, particularly those contracted by immuno-compromised patients in intensive care, the delay introduced by conventional methods can prove fatal to the patient. Even though modified, more convenient, Kirby-Bauer assays have been developed which make use of polymer strips imbibed with an antibiotic concentration gradient and are then applied to agar plate cultures to test not only for absolute susceptibility but also concentration-dependent susceptibility, the time delay issue remains. Commercially available examples of such tests are the E-test (BD Biodisk) or the MICE test (Oxoid).
  • EP2157189 discloses a method and apparatus that allows for susceptibility testing of bacteria via a concentration gradient in a microfluidic device including a microfluidical channel, in which one or more bacteria to be tested are trapped at defined positions on the inner wall of the channel along an antibiotic gradient generated by an antibiotic-loaded hydrogel matrix, and the response to the gradient in antibiotic is measured through fluorescence labeling of dead and live cells.
  • the advantage of such a method is the ability to observe the response of single cells in a reduced time frame in the range of few hours, instead of days required to observe the effects of an antibiotic on a macroscopic scale (e.g. visible inhibition zones on an agar plate).
  • US2013/0196364 discloses a testing method for rapid antibiotic susceptibility testing (RAST) in which a solution of an antibiotic is contacted with a gel matrix comprising immobilized bacteria across a predefined interface in a microfluidic network, such as to allow the diffusion of the solubilized antibiotic into the gel matrix and then measure the response of the bacteria to the antibiotic through an automated image processing system.
  • RAST rapid antibiotic susceptibility testing
  • the present invention provides for a method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics, the method comprising the steps of
  • the present invention further provides a stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics, said assembly comprising a top distribution plate having a plurality of openings, a middle incubation plate having a plurality of wells and a bottom analytic plate having a plurality of wells, wherein
  • the top distribution plate comprises
  • microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate, b. the plurality of wells of the middle incubation plate
  • ii. are each equipped with an element for applying a chemical stimulus or a physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism
  • iii. are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and
  • iv. are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction,
  • the plurality of wells of the bottom analytic plate are optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
  • Fig. 1 shows an disassembled view of the microfluidic assembly comprising a top distribution plate (1) comprising a microfluidic channels (2), a pump (8) and an element for measuring a physical quantity and for applying a physical stimulus (3) reaching into the wells of a middle incubation plate (4) comprising a plurality of wells (9) equipped with an outlet with valve and filtration element (5), a bottom analytic plate (6) comprising a plurality of wells (7).
  • Fig. 2 shows an assembled view of the microfluidic assembly comprising a top distribution plate (1) comprising a microfluidic channels (2), a pump (8) and an element for measuring a physical quantity and for applying a physical stimulus (3) reaching into the wells of the middle incubation plate (4) comprising a plurality of wells (9) equipped with an outlet with valve and filtration element (5), a bottom analytic plate (6).
  • the present invention provides for a method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics, the method comprising the steps of
  • the present invention provides for a method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics.
  • the method according to the present invention is in principle applicable to any kind of microorganism, and is particularly suitable for the detection of the susceptibility of gram- negative bacteria, and in particular bacteria of the Enterobacteriaceae family, such as for example bacteria of the genus Salmonella, Escherichia, Yersinia, Klebsiella, Shigella, Proteus, Enterobacter, Serratia and Citrobacter, Pseudomonas, and Acinetobacter.
  • bacteria of the Enterobacteriaceae family such as for example bacteria of the genus Salmonella, Escherichia, Yersinia, Klebsiella, Shigella, Proteus, Enterobacter, Serratia and Citrobacter, Pseudomonas, and Acinetobacter.
  • the method according to the present invention is in principle applicable to any kind of chemical agents or antibiotics, and is particularly suitable for the detection of the susceptibility of a microorganism to chemical agents such as bactericides and bacteriostats or antibiotics such as those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins) or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides), and in particular ⁇ - lactam antibiotics such as penicillin and its derivatives (penams), cephalosporins (cephems) (e.g.
  • the method according to the present invention comprises a step a. of providing a plurality of liquid microorganism suspension aliquots each having a predetermined volume.
  • the aliquots may be sourced from a liquid microorganism suspension that has been cultured to a predetermined cell concentration; preferably a cell concentration matching 0.5 to 1 McFarland turbidity standard or a cell concentration of 1.2 x 10 8 to 1.8 x 108 CFUs/mL.
  • the microorganisms for culturing a liquid microorganism suspension may be obtained from a medical sample, such as for example blood, urine, mucus, or saliva, or from a sample of soil or water.
  • the liquid microorganism suspension may be a suspension of microorganisms in a suitable liquid culture medium.
  • the culture medium will depend on the type of microorganism, and may be for example Muller-Hinton broth or Lysogeny broth.
  • the aliquots may be provided by dispensing a liquid microorganism suspension into a microtiter plate by either a) pipetting a predetermined volume of liquid microorganism suspension into the microtiter plate with a micropipette or multi-channel micropipette or b) dispensing a predetermined volume of liquid microorganism suspension into the microtiter plate with a microfluidic distribution plate.
  • the microfluidic distribution plate comprises microfluidic channels connected to a dosing pump acting as inlet and a plurality of openings acting as outlets leading into the wells of the microtiter plate.
  • the aliquots may be held in a microtiter plate capable of holding a plurality of aliquots.
  • Known microtiter plates may have 6, 24, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix, and each sample wells may hold an aliquot of from 0.5 to 1.5 ml (24 or 48 wells), 0,1-0,3 ml (96 wells), 0,03-0,1 ml (384 wells).
  • the microtiter plates useful in method according to the present invention are 96 well microtiter plates capable of holding aliquots of 0,1-0,3 ml.
  • the method according to the present invention comprises a step b. of exposing each liquid microorganism suspension aliquot to a chemical stimulus or a physical stimulus.
  • the chemical stimulus may be in the form of a chemical substance at a predetermined concentration such as a phenylpropanoid.
  • the chemical stimulus is a coumarine derivative at a predetermined concentration, such as for example various hydroxycoumarines or 7-azido-4-alkyl-coumarines such as 7-o-2'-(azidomethyl)-benzoyl- 4-methylcoumarin or 7-azido-4-methylcoumarin.
  • Preferred coumarine derivatives are those capable of reacting with endogenous hydrogen sulfide.
  • 7-azido-4- alkylcoumarines react with hydrogen sulfide by forming coumarine.
  • the liquid microorganism suspension aliquots may be exposed to the chemical stimulus at a concentration of from about 0.025 to 0.5 mM, from about 0.025 to 0.125 mM or from about 0.1 to about 0.125 mM.
  • the physical stimulus may be in the form of an electrical potential difference between a pair of electrodes in contact with the liquid microorganism suspension, capable of neutralizing endogenous hydrogen sulfide by oxidizing sulfide ions to elemental sulfur at the anode and reducing protons at the cathode.
  • an electrical potential difference between a pair of electrodes in contact with the liquid microorganism suspension, capable of neutralizing endogenous hydrogen sulfide by oxidizing sulfide ions to elemental sulfur at the anode and reducing protons at the cathode.
  • the method according to the present invention comprises a step c. of incubating each liquid microorganism suspension aliquot in the presence of a different chemical agent or antibiotic to be tested, at varying concentrations, for a predetermined time and at a predetermined temperature.
  • each liquid microorganism suspension aliquot can be incubated in the presence of a different chemical agent or antibiotic, and that thereby the susceptibility of the microorganism to each of the different chemical agents or antibiotics can be tested in a single experimental run. Since the amount of a given chemical agent or antibiotic in each aliquot can be controlled, it is also possible to test the susceptibility of the microorganism to a same chemical agent or antibiotic at varying concentrations. Therefore, the method allows to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIB) for the different chemical agents or antibiotics.
  • MIB minimum bactericidal concentration
  • the ideal incubation temperature is about 37°C, but depending on the microorganism the incubation temperature can be adjusted to a suitable temperature for growth.
  • Each liquid microorganism suspension aliquot may be incubated for about 15 minutes to 120 minutes, preferably from about 15 minutes to 60 min, and more preferably for about 15 minutes to 45 min.
  • the method according to the present invention comprises a step d. of measuring in each liquid microorganism suspension aliquot a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic.
  • the physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic may the current-voltage characteristic of the liquid microorganism suspension. Measuring the current-voltage characteristic allows to measure changes in resistance (or conductivity) in the liquid microorganism suspension aliquot, which changes can be correlated to the response of the microorganism to the chemical agent or antibiotic.
  • the current-voltage characteristic of the liquid microorganism suspension aliquot can be measured with a pair of electrodes in contact with the liquid microorganism suspension and determining the electrical current between the electrodes across the liquid microorganism suspension in dependence to the electrical potential difference between the electrodes.
  • the same pair of electrodes can be used for exposing the aliquots to the physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism in step b. and for measuring the physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic in step d.
  • the method according to the present invention comprises a step e. of separating each liquid microorganism suspension aliquot into its soluble and insoluble fraction.
  • Each liquid microorganism suspension aliquot can be separated into its soluble and insoluble fraction by centrifugation or filtration.
  • the soluble fraction comprises the soluble proteins whereas the insoluble fraction comprises mostly cellular debris such as damaged organelles and cellular membrane and DNA.
  • the soluble fraction corresponds to the supernatant and the insoluble fraction corresponds to the sediments in a centrifuge.
  • the centrifugation is carried out at lO'OOOg or 15'000g for about 5 to 10 minutes at room or incubation temperature.
  • the soluble fraction corresponds to the filtrate and the insoluble fraction to the material retained in the filtering element.
  • the filtration is carried by forcing the liquid microorganism suspension aliquot through the filtering element with positive pressure or vacuum at room or incubation temperature.
  • the method according to the present invention comprises a step f. of collecting the soluble fraction of each aliquot and acquiring a mass spectrum thereof.
  • the mass spectrum can be acquired by a collecting the soluble fraction and mixing the fraction with a suitable solvent such as for example acetonitrile and performing a mass spectrometric analysis, and optionally the fraction with a suitable solvent can be mixed to a carboxylic acid matrix suitable for MALDI mass spectrometry and performing a MALDI mass spectrometric analysis.
  • the method according to the present invention comprises a step g. of collecting the insoluble fraction of each aliquot and acquiring a mass spectrum thereof.
  • the insoluble fraction can be collected by eluting the insoluble fraction from the filtering element with a solvent capable of dissolving the insoluble fraction, preferably a solvent comprising a carboxylic acid, more preferably comprising formic acid or trifluoroacetic acid.
  • the mass spectrum can be acquired by a collecting the insoluble fraction and mixing the fraction with a suitable solvent such as for example acetonitrile and performing a mass spectrometric analysis, and optionally the fraction with a suitable solvent can be mixed to a carboxylic acid matrix suitable for MALDI mass spectrometry and performing a MALDI mass spectrometric analysis.
  • the method according to the present invention comprises a step h. of comparing the acquired mass spectra to reference mass spectra from a database, d
  • the comparison of the acquired mass spectra to reference mass spectra from a database in step h. allows validating the results obtained by measuring the physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic in step d.
  • the present invention further provides for a stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics, said assembly comprising a top distribution plate having a plurality of openings, a middle incubation plate having a plurality of wells and a bottom analytic plate having a plurality of wells, wherein a. the top distribution plate comprises
  • microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate, b. the plurality of wells of the middle incubation plate
  • ii. are each equipped with an element for applying a chemical stimulus or a physical stimulus
  • iii. are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and,
  • iv. are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction,
  • the plurality of wells of the bottom analytic plate are optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
  • the present invention provides for a stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics suitable for carrying out the method described above.
  • the microfluidic distribution plate, middle incubation plate and the bottom analytic plate may be manufactured from various materials such as silicon, glass, ceramics or polymer through processes such as photolithography, machining, embossing or injection moulding or a combination thereof.
  • the top distribution plate serves the purpose of distributing and dosing a plurality of aliquots of predetermined volume into the plurality of wells of the middle incubation plate.
  • the top distribution plate comprises microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate.
  • microfluidic channels of the top distribution plate are connected to a pump acting as inlet, which inlet can be connected to a reservoir of liquid microorganism suspension that has been cultured to a predetermined cell concentration; preferably a cell concentration matching 0.5 to 1 McFarland turbidity standard or a cell concentration of 1.2 x 10 8 to 1.8 x
  • the pump can be controlled to adjust the volume of the aliquots dispensed into each well.
  • the microfluidic channels of the top distribution plate are connected to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate. The position and number of openings acting as outlets will depend on the position and number of the plurality of wells of the middle incubation plate. The geometry of the openings acting as outlets is preferably chosen such that to uncontrolled dripping occurs.
  • the middle incubation plate comprises a plurality of wells, where each of the wells are each loaded with a different chemical agent or antibiotic at different predetermined amounts, each of the wells are each equipped with an element for applying a chemical stimulus or a physical stimulus, each are equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and each are equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction.
  • the middle incubation plate comprises a plurality of wells, which are each loaded with a different chemical agent or antibiotic at different predetermined amounts.
  • the maximum number of different chemical agent or antibiotic to be tested for will depend on the total number of wells in the middle incubation plate.
  • the middle incubation plate may have the same number wells as common microtiter plates having 6, 24, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix, and preferably have 96 or 384 wells.
  • the wells of the middle incubation plate may hold of from 0.5 to 1.5 ml (24 or 48 wells), 0,1-0,3 ml (96 wells), 0,03-0,1 ml (384 wells) of liquid microorganism suspension.
  • the microliter plates useful in method according to the present invention are 96 well microtiter plates capable of holding aliquots of 0,1-0,3 ml.
  • the middle incubation plate may comprise one or more wells void of chemical agent or antibiotic as internal controls.
  • the predetermined amounts of chemical agent or antibiotic loaded in each well will be dependent on the concentration of chemical agent or antibiotic to be achieved once the liquid microorganism suspension is dispensed from the top distribution plate into the wells of the middle incubation plate, and a person of ordinary skill in the art may adjust these parameters without undue burden.
  • the wells may be loaded with the predetermined amount of chemical agent or antibiotic in the form of a dried material or powder, or a concentrated solution.
  • One advantage of the stackable microfluidic assembly is that the middle incubation plate may be prefabricated and then stored until needed.
  • the middle incubation plate comprises a plurality of wells, which are each equipped with an element for applying a chemical stimulus or a physical stimulus.
  • the chemical stimulus may be a predetermined amount of phenylpropanoids, preferably of coumarine or coumarine derivatives such as hydroxycoumarines or 7-azido-4-alkyl-coumarines, more preferably of water-soluble coumarine derivatives.
  • the wells of the middle plate may be loaded with a predetermined amount of chemical stimululus in the same way the wells are loaded with the chemical agent or antibiotic, either in a mixture or separately.
  • the plurality of wells of the middle incubation plate are preferably equipped with a pair of electrodes for applying an electrical potential difference in each well.
  • the electrodes are positioned such as to be in contact with the liquid microorganism suspension when the microfluidic assembly is in use.
  • a pair of electrodes extends from the top distribution plate into each well of the middle plate.
  • a pair of electrodes is incorporated into the wall of each well of the middle plate.
  • the middle incubation plate comprises a plurality of wells, which are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and each are equipped.
  • the element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics can be a pair of electrodes for measuring the current-voltage characteristic and thereby measuring the change in resistance or conductivity.
  • the same pair of electrodes in each well is both the element for applying the stimulus in the wells of the middle plate and the element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms.
  • the same pair of electrodes in each well is both the element for applying the stimulus in the wells of the middle plate and the element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms and the pairs of electrodes extend from the top distribution plate into each well of the middle incubation plate.
  • the middle incubation plate comprises a plurality of wells, which are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the bottom analytic plate.
  • the controllable valve allows retaining the liquid microorganism suspension in the wells of the middle incubation plate for a predetermined time, for example for 15 minutes to 120 minutes, when the micro fluidic assembly is in use.
  • the outlet is equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction.
  • filtering elements include membranes made from materials such as mixed cellulose ester or polyethersulfone (PES) with a filter with pore size of from 0.2 to 0.1 ⁇ .
  • PES polyethersulfone
  • the bottom analytic plate comprises a plurality of wells, optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
  • the number and position of the plurality of wells of the bottom analytic plate will depend on the number and position of the plurality of wells of the middle incubation plate.
  • the microfluidic assembly of the present invention is stackable, i.e. the top distribution plate, the middle incubation plate and the bottom analytic plate may be placed and stacked on top of each other such as to form one assembly that may be handled as one.

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Abstract

A stackable micro fluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics, said assembly comprising a top distribution plate having a plurality of openings, a middle incubation plate having a plurality of wells and a bottom analytic plate having a plurality of wells, wherein a. the top distribution plate comprises i. microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate, b. the plurality of wells of the middle incubation plate i. are each loaded with a different chemical agent or antibiotic (to be tested) at different predetermined amounts, ii. are each equipped with an element for applying a chemical stimulus or a physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism, iii. are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and, iv. are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction, c. the plurality of wells of the bottom analytic plate are optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.

Description

TITLE
Method and microfluidic assembly for antibiotic susceptibility testing
TECHNICAL FIELD The present invention relates to a method and assembly for simultaneous susceptibility testing of microorganisms for different antibiotics and/or chemical agents.
PRIOR ART
The global increase in resistance of bacteria to antimicrobial drugs is generally recognized to be a growing threat for public health. When a patient is infected with an unknown bacteria, it is not only critical to quickly determine with which antibiotic the bacterial infection can be treated most effectively, but also to determine the required concentration of antibiotic to do so. This is expressed by parameters such as the as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIB).
Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo. Testing for antibiotic sensitivity is often done by the well-known Kirby-Bauer method. Small wafers containing antibiotics are placed onto a plate upon which bacteria are growing. If the bacteria are sensitive to the antibiotic, a clear ring, or zone of inhibition, is seen around the wafer indicating poor growth. The inhibition zone diameter can then be correlated to the MIC by taking into account the diffusion rate of the antibiotic through the gel, or by calibrating the assay with susceptibility data measured by, e.g., agar and broth dilution methods. While the Kirby-Bauer method is powerful and versatile, it is a very time consuming process. The method requires several rounds of culturing and incubation, leading to typical test times of 1-3 days depending on the microorganism. This is incompatible with the need for rapid diagnostics and often forces medical practitioners to use initial broad spectrum antibiotic treatment, which is costly, inefficient and increases the problems of antibiotic-resistant bacterial strains in hospital environments. For some infections, particularly those contracted by immuno-compromised patients in intensive care, the delay introduced by conventional methods can prove fatal to the patient. Even though modified, more convenient, Kirby-Bauer assays have been developed which make use of polymer strips imbibed with an antibiotic concentration gradient and are then applied to agar plate cultures to test not only for absolute susceptibility but also concentration-dependent susceptibility, the time delay issue remains. Commercially available examples of such tests are the E-test (BD Biodisk) or the MICE test (Oxoid).
EP2157189 discloses a method and apparatus that allows for susceptibility testing of bacteria via a concentration gradient in a microfluidic device including a microfluidical channel, in which one or more bacteria to be tested are trapped at defined positions on the inner wall of the channel along an antibiotic gradient generated by an antibiotic-loaded hydrogel matrix, and the response to the gradient in antibiotic is measured through fluorescence labeling of dead and live cells. The advantage of such a method is the ability to observe the response of single cells in a reduced time frame in the range of few hours, instead of days required to observe the effects of an antibiotic on a macroscopic scale (e.g. visible inhibition zones on an agar plate).
US2013/0196364 discloses a testing method for rapid antibiotic susceptibility testing (RAST) in which a solution of an antibiotic is contacted with a gel matrix comprising immobilized bacteria across a predefined interface in a microfluidic network, such as to allow the diffusion of the solubilized antibiotic into the gel matrix and then measure the response of the bacteria to the antibiotic through an automated image processing system.
However, the above-cited references suffer from a cost draw-back, since they require the purchase of an image processing system to evaluate the individual cells and their response to a certain antibiotic. There exists thus a need to provide for a reliable method and apparatus that enables for the use of preexisting clinical analytic equipment for the measurement of antibiotic susceptibility, without the need to purchase new analytic equipment. SUMMARY OF THE INVENTION
The present invention provides for a method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics, the method comprising the steps of
a. providing a plurality of liquid microorganism suspension aliquots each having a predetermined volume,
b. exposing each liquid microorganism suspension aliquot to a chemical stimulus or a physical stimulus
c. incubating each liquid microorganism suspension aliquot in the presence of a different chemical agent or antibiotic (to be tested), at varying concentrations, for a predetermined time and at a predetermined temperature,
d. measuring in each liquid microorganism suspension aliquot a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic,
e. separating each liquid microorganism suspension aliquot into its soluble and insoluble fraction,
f. collecting the soluble fraction of each aliquot and acquiring a mass spectrum thereof,
g. collecting the insoluble fraction of each aliquot and acquiring a mass spectrum thereof,
h. comparing the acquired mass spectra to reference mass spectra from a database.
The present invention further provides a stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics, said assembly comprising a top distribution plate having a plurality of openings, a middle incubation plate having a plurality of wells and a bottom analytic plate having a plurality of wells, wherein
a. the top distribution plate comprises
i. microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate, b. the plurality of wells of the middle incubation plate
i. are each loaded with a different chemical agent or antibiotic (to be tested) at different predetermined amounts,
ii. are each equipped with an element for applying a chemical stimulus or a physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism, iii. are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and,
iv. are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction,
c. the plurality of wells of the bottom analytic plate are optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
Further embodiments of the invention are laid down in the dependent claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
Fig. 1 shows an disassembled view of the microfluidic assembly comprising a top distribution plate (1) comprising a microfluidic channels (2), a pump (8) and an element for measuring a physical quantity and for applying a physical stimulus (3) reaching into the wells of a middle incubation plate (4) comprising a plurality of wells (9) equipped with an outlet with valve and filtration element (5), a bottom analytic plate (6) comprising a plurality of wells (7).
Fig. 2 shows an assembled view of the microfluidic assembly comprising a top distribution plate (1) comprising a microfluidic channels (2), a pump (8) and an element for measuring a physical quantity and for applying a physical stimulus (3) reaching into the wells of the middle incubation plate (4) comprising a plurality of wells (9) equipped with an outlet with valve and filtration element (5), a bottom analytic plate (6).
DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides for a method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics, the method comprising the steps of
a. providing a plurality of liquid microorganism suspension aliquots each having a predetermined volume,
b. exposing each liquid microorganism suspension aliquot to a chemical stimulus or a physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism,
c. incubating each liquid microorganism suspension aliquot in the presence of a different chemical agent or antibiotic (to be tested), at varying concentrations, for a predetermined time and at a predetermined temperature, preferably at about 37°C and at for about 15 minutes to 120 minutes,
d. measuring in each liquid microorganism suspension aliquot a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic,
e. separating each liquid microorganism suspension aliquot into its soluble and insoluble fraction,
f. collecting the soluble fraction of each aliquot and acquiring a mass spectrum thereof,
g. collecting the insoluble fraction of each aliquot and acquiring a mass spectrum thereof,
h. comparing the acquired mass spectra to reference mass spectra from a database.
The present invention provides for a method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics.
The method according to the present invention is in principle applicable to any kind of microorganism, and is particularly suitable for the detection of the susceptibility of gram- negative bacteria, and in particular bacteria of the Enterobacteriaceae family, such as for example bacteria of the genus Salmonella, Escherichia, Yersinia, Klebsiella, Shigella, Proteus, Enterobacter, Serratia and Citrobacter, Pseudomonas, and Acinetobacter. The method according to the present invention is in principle applicable to any kind of chemical agents or antibiotics, and is particularly suitable for the detection of the susceptibility of a microorganism to chemical agents such as bactericides and bacteriostats or antibiotics such as those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins) or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides), and in particular β- lactam antibiotics such as penicillin and its derivatives (penams), cephalosporins (cephems) (e.g. ceftriaxon, cefixime), monobactams, and carbapenems (e.g. imipenem, meropenem). The method according to the present invention comprises a step a. of providing a plurality of liquid microorganism suspension aliquots each having a predetermined volume.
The aliquots may be sourced from a liquid microorganism suspension that has been cultured to a predetermined cell concentration; preferably a cell concentration matching 0.5 to 1 McFarland turbidity standard or a cell concentration of 1.2 x 10 8 to 1.8 x 108 CFUs/mL. For example, the microorganisms for culturing a liquid microorganism suspension may be obtained from a medical sample, such as for example blood, urine, mucus, or saliva, or from a sample of soil or water.
The liquid microorganism suspension may be a suspension of microorganisms in a suitable liquid culture medium. The culture medium will depend on the type of microorganism, and may be for example Muller-Hinton broth or Lysogeny broth.
The aliquots may be provided by dispensing a liquid microorganism suspension into a microtiter plate by either a) pipetting a predetermined volume of liquid microorganism suspension into the microtiter plate with a micropipette or multi-channel micropipette or b) dispensing a predetermined volume of liquid microorganism suspension into the microtiter plate with a microfluidic distribution plate. The microfluidic distribution plate comprises microfluidic channels connected to a dosing pump acting as inlet and a plurality of openings acting as outlets leading into the wells of the microtiter plate.
The aliquots may be held in a microtiter plate capable of holding a plurality of aliquots. Known microtiter plates may have 6, 24, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix, and each sample wells may hold an aliquot of from 0.5 to 1.5 ml (24 or 48 wells), 0,1-0,3 ml (96 wells), 0,03-0,1 ml (384 wells). Preferably the microtiter plates useful in method according to the present invention are 96 well microtiter plates capable of holding aliquots of 0,1-0,3 ml. The method according to the present invention comprises a step b. of exposing each liquid microorganism suspension aliquot to a chemical stimulus or a physical stimulus.
The chemical stimulus may be in the form of a chemical substance at a predetermined concentration such as a phenylpropanoid. Preferably the chemical stimulus is a coumarine derivative at a predetermined concentration, such as for example various hydroxycoumarines or 7-azido-4-alkyl-coumarines such as 7-o-2'-(azidomethyl)-benzoyl- 4-methylcoumarin or 7-azido-4-methylcoumarin. Preferred coumarine derivatives are those capable of reacting with endogenous hydrogen sulfide. For example, 7-azido-4- alkylcoumarines react with hydrogen sulfide by forming coumarine. Without wishing to be held to a particular theory, it is believed that the reaction of the microorganism to a chemical agent or antibiotic is accelerated by exposure to the chemical stimulus because of its ability to neutralize endogenous hydrogen sulfide in the microorganism.
The liquid microorganism suspension aliquots may be exposed to the chemical stimulus at a concentration of from about 0.025 to 0.5 mM, from about 0.025 to 0.125 mM or from about 0.1 to about 0.125 mM.
The physical stimulus may be in the form of an electrical potential difference between a pair of electrodes in contact with the liquid microorganism suspension, capable of neutralizing endogenous hydrogen sulfide by oxidizing sulfide ions to elemental sulfur at the anode and reducing protons at the cathode. Without wishing to be held to a particular theory, it is believed that the reaction of the microorganism to a chemical agent or antibiotic is accelerated by exposure to the physical stimulus because of its ability to neutralize endogenous hydrogen sulfide to elemental sulfur in the microorganism.
The method according to the present invention comprises a step c. of incubating each liquid microorganism suspension aliquot in the presence of a different chemical agent or antibiotic to be tested, at varying concentrations, for a predetermined time and at a predetermined temperature.
One advantageous aspect of the method according to the present invention is that each liquid microorganism suspension aliquot can be incubated in the presence of a different chemical agent or antibiotic, and that thereby the susceptibility of the microorganism to each of the different chemical agents or antibiotics can be tested in a single experimental run. Since the amount of a given chemical agent or antibiotic in each aliquot can be controlled, it is also possible to test the susceptibility of the microorganism to a same chemical agent or antibiotic at varying concentrations. Therefore, the method allows to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIB) for the different chemical agents or antibiotics. For most microorganisms and in particular for bacteria of the Enterobacteriaceae family, the ideal incubation temperature is about 37°C, but depending on the microorganism the incubation temperature can be adjusted to a suitable temperature for growth. Each liquid microorganism suspension aliquot may be incubated for about 15 minutes to 120 minutes, preferably from about 15 minutes to 60 min, and more preferably for about 15 minutes to 45 min.
The method according to the present invention comprises a step d. of measuring in each liquid microorganism suspension aliquot a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic.
The physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic may the current-voltage characteristic of the liquid microorganism suspension. Measuring the current-voltage characteristic allows to measure changes in resistance (or conductivity) in the liquid microorganism suspension aliquot, which changes can be correlated to the response of the microorganism to the chemical agent or antibiotic. The current-voltage characteristic of the liquid microorganism suspension aliquot can be measured with a pair of electrodes in contact with the liquid microorganism suspension and determining the electrical current between the electrodes across the liquid microorganism suspension in dependence to the electrical potential difference between the electrodes.
Advantageously, the same pair of electrodes can be used for exposing the aliquots to the physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism in step b. and for measuring the physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic in step d.
The method according to the present invention comprises a step e. of separating each liquid microorganism suspension aliquot into its soluble and insoluble fraction.
Each liquid microorganism suspension aliquot can be separated into its soluble and insoluble fraction by centrifugation or filtration. The soluble fraction comprises the soluble proteins whereas the insoluble fraction comprises mostly cellular debris such as damaged organelles and cellular membrane and DNA.
In the case the liquid microorganism suspension aliquot is separated into its soluble and insoluble fraction by centrifugation, the soluble fraction corresponds to the supernatant and the insoluble fraction corresponds to the sediments in a centrifuge. Preferably, the centrifugation is carried out at lO'OOOg or 15'000g for about 5 to 10 minutes at room or incubation temperature. In the case the liquid microorganism suspension aliquot is separated into its soluble and insoluble fraction by filtration, the soluble fraction corresponds to the filtrate and the insoluble fraction to the material retained in the filtering element. Preferably, the filtration is carried by forcing the liquid microorganism suspension aliquot through the filtering element with positive pressure or vacuum at room or incubation temperature.
The method according to the present invention comprises a step f. of collecting the soluble fraction of each aliquot and acquiring a mass spectrum thereof. The mass spectrum can be acquired by a collecting the soluble fraction and mixing the fraction with a suitable solvent such as for example acetonitrile and performing a mass spectrometric analysis, and optionally the fraction with a suitable solvent can be mixed to a carboxylic acid matrix suitable for MALDI mass spectrometry and performing a MALDI mass spectrometric analysis.
The method according to the present invention comprises a step g. of collecting the insoluble fraction of each aliquot and acquiring a mass spectrum thereof. The insoluble fraction can be collected by eluting the insoluble fraction from the filtering element with a solvent capable of dissolving the insoluble fraction, preferably a solvent comprising a carboxylic acid, more preferably comprising formic acid or trifluoroacetic acid. The mass spectrum can be acquired by a collecting the insoluble fraction and mixing the fraction with a suitable solvent such as for example acetonitrile and performing a mass spectrometric analysis, and optionally the fraction with a suitable solvent can be mixed to a carboxylic acid matrix suitable for MALDI mass spectrometry and performing a MALDI mass spectrometric analysis.
The method according to the present invention comprises a step h. of comparing the acquired mass spectra to reference mass spectra from a database, d
The comparison of the acquired mass spectra to reference mass spectra from a database in step h. allows validating the results obtained by measuring the physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic in step d.
The present invention further provides for a stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics, said assembly comprising a top distribution plate having a plurality of openings, a middle incubation plate having a plurality of wells and a bottom analytic plate having a plurality of wells, wherein a. the top distribution plate comprises
i. microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate, b. the plurality of wells of the middle incubation plate
i. are each loaded with a different chemical agent or antibiotic (to be tested) at different predetermined amounts,
ii. are each equipped with an element for applying a chemical stimulus or a physical stimulus,
iii. are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and,
iv. are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction,
c. the plurality of wells of the bottom analytic plate are optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
The present invention provides for a stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics suitable for carrying out the method described above.
The microfluidic distribution plate, middle incubation plate and the bottom analytic plate may be manufactured from various materials such as silicon, glass, ceramics or polymer through processes such as photolithography, machining, embossing or injection moulding or a combination thereof.
The top distribution plate serves the purpose of distributing and dosing a plurality of aliquots of predetermined volume into the plurality of wells of the middle incubation plate.
The top distribution plate comprises microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate.
The microfluidic channels of the top distribution plate are connected to a pump acting as inlet, which inlet can be connected to a reservoir of liquid microorganism suspension that has been cultured to a predetermined cell concentration; preferably a cell concentration matching 0.5 to 1 McFarland turbidity standard or a cell concentration of 1.2 x 108 to 1.8 x
10 CFUs/mL. The pump can be controlled to adjust the volume of the aliquots dispensed into each well. The microfluidic channels of the top distribution plate are connected to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate. The position and number of openings acting as outlets will depend on the position and number of the plurality of wells of the middle incubation plate. The geometry of the openings acting as outlets is preferably chosen such that to uncontrolled dripping occurs.
The middle incubation plate comprises a plurality of wells, where each of the wells are each loaded with a different chemical agent or antibiotic at different predetermined amounts, each of the wells are each equipped with an element for applying a chemical stimulus or a physical stimulus, each are equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and each are equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction.
The middle incubation plate comprises a plurality of wells, which are each loaded with a different chemical agent or antibiotic at different predetermined amounts. The maximum number of different chemical agent or antibiotic to be tested for will depend on the total number of wells in the middle incubation plate. The middle incubation plate may have the same number wells as common microtiter plates having 6, 24, 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix, and preferably have 96 or 384 wells. The wells of the middle incubation plate may hold of from 0.5 to 1.5 ml (24 or 48 wells), 0,1-0,3 ml (96 wells), 0,03-0,1 ml (384 wells) of liquid microorganism suspension. Preferably the microliter plates useful in method according to the present invention are 96 well microtiter plates capable of holding aliquots of 0,1-0,3 ml. Optionally, the middle incubation plate may comprise one or more wells void of chemical agent or antibiotic as internal controls. The predetermined amounts of chemical agent or antibiotic loaded in each well will be dependent on the concentration of chemical agent or antibiotic to be achieved once the liquid microorganism suspension is dispensed from the top distribution plate into the wells of the middle incubation plate, and a person of ordinary skill in the art may adjust these parameters without undue burden. The wells may be loaded with the predetermined amount of chemical agent or antibiotic in the form of a dried material or powder, or a concentrated solution. One advantage of the stackable microfluidic assembly is that the middle incubation plate may be prefabricated and then stored until needed.
The middle incubation plate comprises a plurality of wells, which are each equipped with an element for applying a chemical stimulus or a physical stimulus.
In the case where the plurality of wells are each equipped with an element for applying a chemical stimulus, the chemical stimulus may be a predetermined amount of phenylpropanoids, preferably of coumarine or coumarine derivatives such as hydroxycoumarines or 7-azido-4-alkyl-coumarines, more preferably of water-soluble coumarine derivatives. The wells of the middle plate may be loaded with a predetermined amount of chemical stimululus in the same way the wells are loaded with the chemical agent or antibiotic, either in a mixture or separately. In the case where the plurality of wells are each equipped with an element for applying a physical stimulus, the plurality of wells of the middle incubation plate are preferably equipped with a pair of electrodes for applying an electrical potential difference in each well. The electrodes are positioned such as to be in contact with the liquid microorganism suspension when the microfluidic assembly is in use. In one embodiment, a pair of electrodes extends from the top distribution plate into each well of the middle plate. In another embodiment, a pair of electrodes is incorporated into the wall of each well of the middle plate. The middle incubation plate comprises a plurality of wells, which are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and each are equipped. The element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics can be a pair of electrodes for measuring the current-voltage characteristic and thereby measuring the change in resistance or conductivity. In a preferred embodiment, the same pair of electrodes in each well is both the element for applying the stimulus in the wells of the middle plate and the element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms.
In another preferred embodiment, the same pair of electrodes in each well is both the element for applying the stimulus in the wells of the middle plate and the element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms and the pairs of electrodes extend from the top distribution plate into each well of the middle incubation plate. The middle incubation plate comprises a plurality of wells, which are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the bottom analytic plate. The controllable valve allows retaining the liquid microorganism suspension in the wells of the middle incubation plate for a predetermined time, for example for 15 minutes to 120 minutes, when the micro fluidic assembly is in use. The outlet is equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction. Exemplary filtering elements include membranes made from materials such as mixed cellulose ester or polyethersulfone (PES) with a filter with pore size of from 0.2 to 0.1 μηι. The bottom analytic plate comprises a plurality of wells, optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
The number and position of the plurality of wells of the bottom analytic plate will depend on the number and position of the plurality of wells of the middle incubation plate.
The microfluidic assembly of the present invention is stackable, i.e. the top distribution plate, the middle incubation plate and the bottom analytic plate may be placed and stacked on top of each other such as to form one assembly that may be handled as one.
LIST OF REFERENCE SIGNS
1 top distribution plate
2 microfluidic channels
3 element for measuring a physical quantity and applying a physical stimulus
4 middle incubation plate
5 outlet with controllable valve and filtering element
6 bottom analytic plate
7 well
8 pump
9 well

Claims

1. A method for simultaneously determining the susceptibility of a microorganism to different chemical agents or antibiotics, the method comprising the steps of
a. providing a plurality of liquid microorganism suspension aliquots each having a predetermined volume,
b. exposing each liquid microorganism suspension aliquot to a chemical stimulus or a physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism,
c. incubating each liquid microorganism suspension aliquot in the presence of a different chemical agent or antibiotic (to be tested), at varying concentrations, for a predetermined time and at a predetermined temperature, preferably at about 37°C and at for about 15 minutes to 120 minutes,
d. measuring in each liquid microorganism suspension aliquot a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic,
e. separating each liquid microorganism suspension aliquot into its soluble and insoluble fraction,
f. collecting the soluble fraction of each aliquot and acquiring a mass spectrum thereof,
g. collecting the insoluble fraction of each aliquot and acquiring a mass spectrum thereof,
h. comparing the acquired mass spectra to reference mass spectra from a database.
2. The method according to claim 1, wherein the mass spectra are acquired by MALDI mass spectrometry.
3. The method according to claim 1 or 2 wherein each liquid microorganism suspension aliquot is separated into its soluble and insoluble fraction by centrifugation or filtration, preferably by filtration through a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction.
4. The method according to claim 3 wherein the insoluble fraction is collected by eluting the insoluble fraction from the filtering means with a solvent capable of dissolving the insoluble fraction, preferably a solvent comprising a carboxylic acid, more preferably comprising formic acid.
5. The method according to any one of claims 1 to 4, wherein the liquid microorganism suspension aliquots have a predetermined microorganism concentration, preferably a concentration matching 0.5 to 1 McFarland turbidity standard.
6. The method according to any one of claims 1 to 5, wherein the microorganisms are gram-negative bacteria, preferably bacteria of the Enterobacteriaceae family.
7. The method according to any one of claims 1 to 6, wherein each liquid microorganism suspension aliquot is exposed to a physical stimulus.
8. The method according to claim 7, wherein the physical stimulus is an electrical potential difference between a pair of electrodes in contact with the liquid microorganism suspension.
9. The method according to any one of claims 1 to 6, wherein each liquid microorganism suspension aliquot is exposed to a chemical stimulus.
10. The method according to claim 9, wherein the chemical stimulus is a phenylpropanoid at a predetermined concentration, preferably a coumarine or coumarine derivative such as a hydroxycoumarine or 7-azido-4-alkyl-coumarine.
11. The method according to any one of claims 1 to 10, wherein the physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agent or antibiotic is the current-voltage characteristic of the liquid microorganism suspension.
12. A stackable microfluidic assembly for simultaneously determining the susceptibility of microorganisms to different chemical agents or antibiotics, said assembly comprising a top distribution plate having a plurality of openings, a middle incubation plate having a plurality of wells and a bottom analytic plate having a plurality of wells, wherein
a. the top distribution plate comprises
i. microfluidic channels connected to a pump acting as inlet and to a plurality of openings acting as outlets leading into the plurality of wells of the middle incubation plate, b. the plurality of wells of the middle incubation plate
i. are each loaded with a different chemical agent or antibiotic (to be tested) at different predetermined amounts,
ii. are each equipped with an element for applying a chemical stimulus or a physical stimulus capable of neutralizing hydrogen sulphide endogenous to the microorganism,
iii. are each equipped with an element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics and,
iv. are each equipped with a controllable valve for controlling the flow through an outlet leading into the corresponding well in the analytic plate, said outlet being equipped with a filtering element suitable for separating a liquid microorganism suspension into its soluble and insoluble fraction,
c. the plurality of wells of the bottom analytic plate are optionally loaded with a carboxylic acid matrix solution suitable for MALDI mass spectrometry.
13. The method according to claim 12, wherein the plurality of wells of the middle incubation plate are equipped with an element for applying a physical stimulus, preferably a pair of electrodes for applying an electrical potential difference in each well.
14. The method according to claim 12, wherein the plurality of wells of the middle incubation plate are equipped with an element for applying a chemical stimulus, preferably a predetermined amount of phenylpropanoids, preferably of coumarine or coumarine derivatives such as hydroxycoumarines or 7-azido-4-alkyl- coumarines, more preferably of water-soluble coumarine derivatives.
15. The method according to claim 14, wherein the hydroxycoumarines are chosen from 7-hydroxycoumarine such as umbelliferone and alkyl esters thereof; 4- hydroxycoumarine and derivatives thereof having a substituent at the 3 -position such as acenocoumarol, dicoumarol, ethyl biscoumacetate, phenprocoumon, warfarin, coumatetralyl, difenacoum, flocoumafen, bromadiolone, tioclomarol or brodifacoum; 6,7-dihydroxycoumarine such as aesculetin and alkyl esters thereof.
16. The method according to any one of claims 12 to 15, wherein the element for measuring a physical quantity suitable for determining the susceptibility of the microorganisms to the chemical agents or antibiotics is a pair of electrodes for measuring a current-voltage characteristic.
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