Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/18—Testing for antimicrobial activity of a material
• • 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
  • C12Q1/06—Quantitative determination

Definitions

• the invention relates to the field of biological diagnostics, in particular biomedical diagnostics in the context of researching resistance to antimicrobial agents.
• the current problem which is worldwide, mainly concerns Gram-negative bacteria such as Enterobacteriaceae, Pseudomonas aeruginosa or Acinetobacter baumannii , even if problems remain with regard to certain Gram-positive bacteria such as Staphylococcus , Enterococcus , etc.
• the present inventors propose to use the technique of Fractionation by Flux-Force coupling (Field Flow Fractionation or FFF) developed in the late 1960s by J.C. Giddings.
• FFF Field Flow Fractionation
• This method is often presented as one of the most versatile separation methods. Indeed, the great variety of usable fields (hydrodynamics, gravity, electric, magnetic, ...), of instrumental configurations, of elution modes, allow to consider an infinity of experimental conditions to be implemented for sorting, separation. and the characterization of polymers, powders, emulsions, colloids, nanoparticles or bioparticles: macromolecules, viruses, organelles and cells whose size varies between 10 nm and 100 ⁇ m.
• SdFFF uses a multigravitational field and remains one of the most widely used FFF methods.
• This method has a high sensitivity in terms of change in size, density, shape or deformability of the analyzed objects, and allows early recording of metabolic changes in the cell population without specific preparation of the analyzed sample. It is thus possible, by simple comparison of elution profiles or fractograms, to demonstrate a metabolic modification between an untreated control analytical sample and an analytical sample subjected to the biological event.
• a subject of the present invention is thus a method for determining the resistance of a microorganism to at least one antimicrobial, characterized in that the method comprises the steps of:
• the constituent microorganism of the microbial population can be any one selected from bacteria, fungi, yeasts, protozoa.
• the invention is not limited to a given resistance mechanism, nor to a given species of microorganism. It does not require a heavy inoculum but an inoculum comparable to that used by the usual methods in bacteriology.
• the antimicrobial can be any one selected from antibiotics, antifungals, antiparasitic agents.
• the microorganism is a bacterium.
• bacterium is understood to mean any type of bacterium, whether of the Gram-negative type or of the Gram-positive type.
• the antimicrobial is an antibiotic.
• antibiotic means a natural or synthetic organic substance which exerts its action on bacteria by destroying them (bactericidal effect) or by inhibiting their growth and multiplication (bacteriostatic effect).
• the microbial population can be derived from a biological sample.
• the biological sample can also be of human or animal origin; it can also be of environmental origin.
• the biological sample may be a sample of urine, stool, sputum, pus, cerebrospinal fluid, blood or the like.
• the biological sample can be a reference strain.
• reference strain is understood to mean an isolated strain of microorganisms whose characters have been studied and which is kept over a long period in a bank of reference strains.
• biological samples within the meaning of the present invention.
• the microbial population as understood in the present invention can thus be a microbial suspension obtained from a mixture of visually identical colonies isolated from a biological sample.
• This suspension is then incubated in the presence of antimicrobials of various kinds at different concentrations (treated) or incubated for the same duration in the absence of antimicrobial (control). These samples are the “analytical samples” which will be eluted in the flow-force coupling fractionation device.
• the incubation step with the antimicrobial may have a duration ranging from 30 to 120 minutes, preferably from 30 to 60 minutes.
• the incubation step with the antimicrobial is performed in a liquid culture medium.
• the flow-force coupling fractionation device used in the method of the invention can be a flow-force coupling fractionation device of multigravitational or centrifugal, hydrodynamic, dielectrophoretic (DEP), electrical, magnetic, thermal, or other type. any other type of suitable flow-force coupling fractionation device.
• DEP dielectrophoretic
• FFF hydrodynamic FFF
• asymmetrical channel or asymmetric (Asymetrical Flow FFF) and where only the accumulation wall consists of a semi-permeable membrane.
• Magnetic FFF Magnetic FFF
• the flow-force coupling fractionation device is a multigravitational type flow-force coupling fractionation device.
• the non-retained species correspond to molecules originating from the culture medium, to biological or cellular debris, etc., which will absorb the UV signal from the detector, but which are not sensitive to the field used.
• molecules of size less than ⁇ m do not undergo the effect of the field: they are therefore not retained. They move through the system at the same speed as the mobile phase, that is, the liquid vector in the device.
• this is the time required for the mobile phase to travel through the flow-force coupling fractionation device from the injection system (valve Rheodyne® type) to the detector (UV-Vis spectrophotometer type) through the tubes and the separation channel inserted in the centrifugation bowl.
• the “threshold of significance” is the threshold above which the microbial population is considered to be susceptible or resistant to the antimicrobial. Its value is defined for each microorganism / antimicrobial pair, as a function of the usual active doses of this antimicrobial. The values will be referenced in a dedicated database.
• the micro-organism is considered to be resistant. If the measured value of P ⁇ R> at the significance level, then the micro-organism is considered to be sensitive.
• the degree of sensitivity of the detection method makes it possible to demonstrate “sensitive” and “resistant” profiles to an antimicrobial.
• P ⁇ R present for reference strains of E. coli the following values:
• kits one or more solutions for cleaning and decontaminating the components in order to reduce the ecological impact and allow their reuse. It is envisioned that at least two sets of rolling kits can be used, one in use, the other in cleaning and decontamination. Also, used kits can be recovered by the manufacturer to ensure their recycling and reconditioning, always with a view to reducing the ecological and economic impact.
• separation channel means the flow channel to which an external field of variable nature is applied depending on the type of FFF used and in which the analyzed microorganisms circulate.
• the walls of the separation channel vary depending on the type of FFF.
• the walls are non-permeable and rigid. This type of wall is shared with the MgFFF.
• the walls are non-permeable plates which are heated to different temperatures.
• the ElFFF these are electrodes.
• at least one of the two walls is a wall associated with a semi-permeable membrane and consists of a frit.
• rotary joints is understood to mean the device which allows the passage of the mobile phase and the samples at the inlet and at the outlet of the separation channel from a fixed reference frame (namely mobile phase pump, sample injector, detector. sample) to a rotating frame (the separation channel). This passage must be done without leakage of liquid and therefore of risk of dispersion of the sample, and the potential contamination of the operator.
• Rotary joints are strategic parts in a SdFFF device.
• tubing is meant the pipes bringing the samples from the sample injector to the separation channel as well as those bringing the separated species from the separation channel to the detector.
• the detector differs depending on the type of FFF technique.
• the most commonly used is a UV-Vis spectrophotometer type detector allowing the measurement of the variation in absorbance over time.
• the present invention relates in particular to a kit for determining the resistance of a bacterial population to an antibiotic by a SdFFF fractionation technique comprising:
• Example 2 represents the analytical scheme for measuring resistance to an antibiotic according to the implementation of Example 2.
• the strain is very sensitive to the antimicrobial.
• the strain is resistant to the antimicrobial.
• the elution profiles of the reference strain E.coli ATCC EC 25922, incubated in the absence (control) or in the presence of increasing doses of ampicillin ( 4 or 8 mg / mL), then it can be seen that, unlike the strain E.coli ATCC EC 35218, E.coli 25922 is sensitive to ampicillin. Indeed, the retention times (tr) for the control conditions (on average 2.66 min) and treated with 4 mg / mL of ampicillin (2.36 min on average) allowing Robs' calculations for the control (0.362 ) and the treated samples (0.408 for 4 mg / ml), which corresponds to a percentage variation of the retention factor P ⁇ R 12.70; 10% can be considered as the significance threshold. At this concentration, the strain is sensitive to the antimicrobial.
• the flow-force coupling fractionation device is established in accordance with patent EP1679124.
• the device comprises an injection system where the sample is taken care of, the tubing bringing the sample from the injector to the separation channel, via a first rotating joint, the separation channel contained in a centrifugation bowl of which the rotation is ensured by an electric motor itself controlled manually or via a suitable computer device, thus allowing the control of the intensity of the multigravitational field, the tubing bringing the separated species from the channel to the detector, via the second rotating joint, and finally the detector.
• Bacterial strains used E.coli 25922 and 35218 (ATCC, Manassas, VA, USA).
• Antibiotics Ampicillin (Sigma-Aldrich, Saint-Quentin-Fallavier, France, ref. 21442020), Kanamycin (MP Biomedical, Illkirch FRANCE, ref. 150020) Chlortetracycline (Sigma-Aldrich, Saint-Quentin-Fallavier, France, Ref 17776) .
• MH Mueller-Hinton
• the culture medium was centrifuged and then taken up in a volume of 0.5 mL of PBS.
• Table 1 Results of the analysis of the fractograms according to the experiment shown in Figures 5A and 5B: calculation of the P ⁇ R allowing, by comparison with the significance threshold, set at 10% for these strains and this antibiotic, the determination of the resistance of E.coli strains 25922 and 35218 with respect to Ampicilin
• Example 1 Results of tests of resistance of the strain E.coli ATCC 25922 to various molecules by means of the SdFFF according to the present invention.
• the SdFFF elution conditions were adapted from those commonly used in cell sorting practices for eukaryotic cells.
• the direct introduction of the sample in the direction opposite to the gravity field, allowing a rapid relaxation of the species in the effective flow lines for their separation is here optimized by a short additional stop-flow step, due to the small sizes ( ⁇ 2 ⁇ m) of the species to be separated.
• the detection method of the present invention allows a quantitative, dose-dependent response, which is complementary to its qualitative dimension, namely the measurement of the threshold of significance of resistance vs. antimicrobial sensitivity.
• the sensitivity of the detection process depends on the ability of the FFF apparatus to record changes in retention depending on the concentration of antimicrobial used.
• Example 2 A new analysis protocol was established according to the , faithful to the clinical implementation of conventional antibiograms, making it possible to reduce the obtaining of test results to 26 hours after collection / isolation.
• an antibiogram is carried out using bacterial colonies obtained from a biological sample.
• the time to obtain these colonies is on average 16-24 hours, but it can sometimes be shorter, in the order of 10-12 hours depending on the bacterial species.
• These colonies are then resuspended in liquid medium and incubated for 16-24 hours with different antibiotics according to the recommendations of the CA-SFM (Committee of the Antibiogram of the French Society of Microbiology) in force.
• the difference in behavior of the bacterial strain is demonstrated by measuring R obs , calculating the P ⁇ R and comparing it with thresholds of significance making it possible to indicate the sensitivity or resistance of the bacterial strain to the action. bactericidal or bacteriostatic of the tested antibiotic.
• Antimicrobial resistance is measured by comparing P ⁇ R to the significance level, P ⁇ R is expressed according to the following formula:
• the inventive nature of the present invention lies in the simplicity and speed of its implementation.
• the method has the advantage of allowing the automation of the steps, from the automatic injection of the samples into the device until the result in terms of sensitive / resistant is obtained.
• the simplicity and speed of the method of the invention is based on the fact that it is completely devoid of pre-treatment of the sample to be eluted, resulting in faster results.
• the method of the invention further has an aspect of universality and is applicable to any type of microorganism, namely bacteria, fungi, yeasts, protozoa. It is not necessary to first identify a specific antigen that should allow the separation of microbial populations as required with flow cytometry.

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• 2020
  • 2020-04-06 FR FR2003429A patent/FR3108917B1/fr active Active
• 2021
  • 2021-04-06 WO PCT/IB2021/052832 patent/WO2021205330A1/fr unknown
  • 2021-04-06 CA CA3173718A patent/CA3173718A1/fr active Pending
  • 2021-04-06 US US17/917,231 patent/US20230167479A1/en active Pending
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